36P6D5: secreted tumor antigen

ABSTRACT

Described is a gene and its encoded secreted tumor antigen, termed 36P6D5, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers which express 36P6D5, particularly including cancers of the bladder, kidney, prostate, breast, colon, ovary and pancreas.

The present application is a divisional application of U.S. patentapplication Ser. No. 10/365,254, filed Feb. 11, 2003, now U.S. Pat. No.7,223,542, which is a divisional of U.S. patent application Ser. No.09/702,114, filed on Oct. 30, 2000, now U.S. Pat. No. 6,566,078, whichclaims the benefit of priority of U.S. Provisional Application No.60/162,417 filed Oct. 28, 1999. The contents of the above patentapplications are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention described herein relates to a gene and its encoded tumorantigen, termed 36P6D5, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers that express36P6D5 gene products.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text file as follows:

File Name Date of Creation Size (bytes) 511582002211seqlist.txt Sep. 18,2007 10,016 bytes

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people each year, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Many cancer patients experience a recurrence.

Generally speaking, the fundamental problem in the management of thedeadliest cancers is the lack of effective and non-toxic systemictherapies. Molecular medicine, still very much in its infancy, promisesto redefine the ways in which these cancers are managed. Unquestionably,there is an intensive worldwide effort aimed at the development of novelmolecular approaches to cancer diagnosis and treatment. For example,there is a great interest in identifying truly tumor-specific genes andproteins that could be used as diagnostic and prognostic markers and/ortherapeutic targets or agents. Research efforts in these areas areencouraging, and the increasing availability of useful moleculartechnologies has accelerated the acquisition of meaningful knowledgeabout cancer. Nevertheless, progress is slow and generally uneven.

As discussed below, the management of prostate cancer serves as a goodexample of the limited extent to which molecular biology has translatedinto real progress in the clinic. With limited exceptions, the situationis more or less the same for the other major carcinomas mentioned above.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy remain fixed as the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with significant undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the management of this disease. Although the serum PSAassay has been a very useful tool, its specificity and general utilityis widely regarded as lacking in several important respects, as furtherdiscussed below. Most prostate cancers initially occur in the peripheralzone of the prostate gland, away from the urethra. Tumors within thiszone may not produce any symptoms and, as a result, most men withearly-stage prostate cancer will not present clinical symptoms of thedisease until significant progression has occurred. Tumor progressioninto the transition zone of the prostate may lead to urethralobstruction, thus producing the first symptoms of the disease. However,these clinical symptoms are indistinguishable from the commonnon-malignant condition of benign prostatic hyperplasia (BPH). Earlydetection and diagnosis of prostate cancer currently relies on digitalrectal examinations (DRE), prostate specific antigen (PSA) measurements,transrectal ultrasonography (TRUS), and transrectal needle biopsy(TRNB). At present, serum PSA measurement in combination with DRErepresent the leading tool used to detect and diagnose prostate cancer.Both have major limitations which have fueled intensive research intofinding better diagnostic markers of this disease.

Similarly, there is no available marker that can predict the emergenceof the typically fatal metastatic stage of prostate cancer. Diagnosis ofmetastatic stage is presently achieved by open surgical or laparoscopicpelvic lymphadenectomy, whole body radionuclide scans, skeletalradiography, and/or bone lesion biopsy analysis. Clearly, better imagingand other less invasive diagnostic methods offer the promise of easingthe difficulty those procedures place on a patient, as well as improvingdiagnostic accuracy and opening therapeutic options. A similar problemis the lack of an effective prognostic marker for determining whichcancers are indolent and which ones are or will be aggressive. PSA, forexample, fails to discriminate accurately between indolent andaggressive cancers. Until there are prostate tumor markers capable ofreliably identifying early-stage disease, predicting susceptibility tometastasis, and precisely imaging tumors, the management of prostatecancer will continue to be extremely difficult.

PSA is the most widely used tumor marker for screening, diagnosis, andmonitoring prostate cancer today. In particular, several immunoassaysfor the detection of serum PSA are in widespread clinical use. Recently,a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for PSAmRNA in serum has been developed. However, PSA is not a disease-specificmarker, as elevated levels of PSA are detectable in a large percentageof patients with BPH and prostatitis (25-86%) (Gao et al., 1997,Prostate 31: 264-281), as well as in other nonmalignant disorders and insome normal men, a factor which significantly limits the diagnosticspecificity of this marker. For example, elevations in serum PSA ofbetween 4 to 10 ng/ml are observed in BPH, and even higher values areobserved in prostatitis, particularly acute prostatitis. BPH is anextremely common condition in men. Further confusing the situation isthe fact that serum PSA elevations may be observed without anyindication of disease from DRE, and visa-versa. Moreover, it is nowrecognized that PSA is not prostate-specific (Gao et al., supra, forreview).

Various methods designed to improve the specificity of PSA-baseddetection have been described, such as measuring PSA density and theratio of free vs. complexed PSA. However, none of these methodologieshave been able to reproducibly distinguish benign from malignantprostate disease. In addition, PSA diagnostics have sensitivities ofbetween 57-79% (Cupp & Osterling, 1993, Mayo Clin Proc 68:297-306), andthus miss identifying prostate cancer in a significant population of menwith the disease.

There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter et al., 1996, Proc.Natl. Acad. Sci. USA 93: 749; Bzdega et al., 1997, J. Neurochem. 69:2270). However, the expression of PSM in small intestine and brain(Israeli et al., 1994, Cancer Res. 54: 1807), as well its potential rolein neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med 21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252) andprostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl.Acad. Sci. USA 95: 1735). PCTA-1, a novel galectin, is largely secretedinto the media of expressing cells and may hold promise as a diagnosticserum marker for prostate cancer (Su et al., 1996). PSCA, a GPI-linkedcell surface molecule, was cloned from LAPC-4 cDNA and is unique in thatit is expressed primarily in basal cells of normal prostate tissue andin cancer epithelia (Reiter et al., 1998). Vaccines for prostate cancerare also being actively explored with a variety of antigens, includingPSM and PSA.

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

SUMMARY OF THE INVENTION

The present invention relates to a gene and protein designated 36P6D5.In normal individuals, 36P6D5 protein appears to be predominantlyexpressed in pancreas, with lower levels of expression detected inprostate and small intestine. The 36P6D5 gene is also expressed inseveral human cancer xenografts and cell lines derived from prostate,breast, ovarian and colon cancers, in some cases at high levels.Over-expression of 36P6D5, relative to normal, is observed in prostatecancer xenografts initially derived from a prostate cancer lymph nodemetastasis and passaged intratibially and subcutaneously in SCID mice.Extremely high level expression of 36P6D5 is detected in the breastcancer cell line DU4475, a cell line that was initially derived from amammary gland carcinoma (Langlois et al., 1979, Cancer Res. 39: 2604).The 36P6D5 gene is also expressed in tumor patient samples derived frombladder, kidney, colon and lung cancers, in some cases at high levels.

A full length 36P6D5 cDNA of 931 bp (SEQ ID NO: 1) provided hereinencodes a 235 amino acid open reading frame (SEQ ID NO: 2) withsignificant homology to the 2-19 protein precursor (Genbank P98173) aswell as a gene previously cloned from human osteoblasts (Q92520). Thepredicted 235 amino acid 36P6D5 protein also contains an N-terminalsignal sequence, indicating that the 36P6D5 protein is secreted. The36P6D5 gene therefore encodes a secreted tumor antigen which may beuseful as a diagnostic, staging and/or prognostic market for, and/or mayserve as a target for various approaches to the treatment of, prostate,breast, colon, pancreatic, and ovarian cancers expressing 36P6D5. Thepredicted molecular weight of the 36P6D5 protein is approximately 26 kDand its' pI is 8.97.

Expression analysis demonstrates high levels of 36P6D5 expression inseveral prostate and other cancer cell lines as well as prostate cancerpatient samples and tumor xenografts. The expression profile of 36P6D5in normal adult tissues, combined with the over-expression observed incancer cells such as bladder, colon, kidney, breast, lung, ovary andprostate cancer cell lines and/or cancer patient samples, providesevidence that 36P6D5 is aberrantly expressed in at least some cancers,and can serve as a useful diagnostic and/or therapeutic target for suchcancers.

The invention provides polynucleotides corresponding or complementary toall or part of the 36P6D5 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding 36P6D5proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and relatedmolecules, polynucleotides or oligonucleotides complementary to the36P6D5 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 36P6D5 genes, mRNAs, or to36P6D5-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 36P6D5. Recombinant DNA moleculescontaining 36P6D5 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 36P6D5gene products are also provided. The invention further provides 36P6D5proteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to 36P6D5 proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, and antibodies labeled with a detectablemarker.

The invention further provides methods for detecting the presence andstatus of 36P6D5 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 36P6D5. Atypical embodiment of this invention provides methods for monitoring36P6D5 gene products in a tissue sample having or suspected of havingsome form of growth dysregulation such as cancer.

The invention further provides various therapeutic compositions andstrategies for treating cancers that express 36P6D5 such as prostatecancers, including therapies aimed at inhibiting the transcription,translation, processing or function of 36P6D5 as well as cancervaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ IDNO: 2) sequences of 36P6D5 cDNA. The start methionine and consensusKozak sequence are indicated in bold and the putative N-terminal signalsequence is underlined.

FIGS. 2A-2B. Amino acid sequence alignment of the 36P6D5 ORF with 2-19protein precursor (2A) (SEQ ID NO: 15) and GS3786 protein (osteoblastprotein) (2B) (SEQ ID NO: 16). Percent sequence identities are indicatedon the figure.

FIG. 3. Northern blot analysis of human 36P6D5 expression in variousnormal tissues showing predominant expression in pancreas and low levelexpression in prostate and small intestine.

FIGS. 4A-4B. Northern blot analysis of human 36P6D5 mRNA expression in apanel of prostate cancer xenografts and various other human cancer celllines.

FIGS. 5A-5B. Secretion of 36P6D5 protein from transfected 293T cells anddetection with anti-36P6D5 polyclonal antibody. 293T cells weretransiently transfected with either pCDNA 3.1 MYC/HIS 36P6D5, pTag536P6D5, in which the natural signal sequence of 36P6D5 is replaced withan immunoglobulin signal sequence, or pAPTag5 36P6D5 which encodes afusion protein composed of 36P6D5 and alkaline phosphatase (alsocontaining the Ig signal sequence). Conditioned media or whole celllysates were subjected to Western blotting with either rabbit anti-HispAb (Santa Cruz, Biotechnology, Inc., 1:2,500 dilution, left panel) orwith affinity purified rabbit anti-36P6D5 pAb (1 ug/ml, right panel).Anti-His and anti-36P6D5 pAb reactive bands were visualized byincubation of the blots with anti-rabbit-HRP conjugated secondaryantibody followed by enhanced chemiluminescence detection.

FIG. 6. Expression of 36P6D5 in cancer patient tumors. RT-PCR analysisof 36P6D5 mRNA expression in bladder cancer, kidney cancer, coloncancer, and lung cancer patient tumors.

FIG. 7. 36P6D5 expression in bladder cancer and their matched normaltissues was tested by Northern blot analysis. For this figure, 10 μg oftotal RNA were loaded for each sample. Overexpression of 36P6D5expression was detected in 3 out of 4 cancers tested lanes 3, 5, 7 and8). No expression was seen in bladder tissue isolated from a normalindividual lane 1).

FIG. 8. Binding of 36P6D5 to LAPC9 AD. A single cell suspension of LAPC9AD xenograft cells were allowed to adhere overnight to a 6 well plate.The cells were incubated in the presence of control or 36P6D5-AP fusionprotein. The alkaline phosphate substrate, BM purple, was used fordetection.

FIGS. 9A-9B. Human cancer cells express and secrete 36P6D5 protein.Conditioned media and/or cell lysates from a variety of cancer celllines representing cancers derived from prostate (LAPC4 xenograft),colon (Colo 205, CaCo-1), breast (Du4475), and pancreatic (Capan-1)tissues, as well as PC3 prostate cancer cells engineered to overexpress36P6D5 protein, were subjected to Western analysis using an anti-36P6D5murine pAb. The specific anti-36P6D5 immunoreactive bands representingendogenous 36P6D5 protein are indicated with arrows and runapproximately between 35 and 40 kD. The molecular weight of 36P6D5calculated from the amino acid sequence is 26 kD suggesting thatendogenous 36P6D5 protein is post-translationally modified, possibly byglycosylation.

FIG. 10. A sensitive and specific capture ELISA detects 36P6D5 proteinin supernatants of human cancer cell lines. A capture ELISA wasdeveloped using protein G purified murine anti-36P6D5 pAb as capture Aband a biotinylated form of the same pAb as detection Ab. Shown is thestandard curve generated using the Tag5-36P6D5 protein and specificdetection and quantitation of 36P6D5 present in supernatants derivedfrom PC-3-Neo transfected cells (O.D.=0.023, 36P6D5 proteinconcentration in ng/ml=N.D.), PC-3 cells overexpressing 36P6D5(O.D.=0.186, 36P6D5 protein concentration in ng/ml=1.48) and endogenous36P6D5 protein secreted by Du4475 breast cancer cells (O.D.=0.085,36P6D5 protein concentration in ng/ml=0.50).

FIGS. 11A-11B. Detection of 36P6D5 expression in human cancers. Celllysates from Colon, breast and kidney cancer tissues (Ca), as well astheir normal matched adjacent tissues (N) were subjected to Westernanalysis using an anti-36P6D antibody. The specific anti-36P6D5immunoreactive bands represent a monomeric form of the 36P6D5 protein,which runs approximately between 35 and 40 kD, and multimeric forms ofthe protein, which run approximately at 90 and 120 kD.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers which have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers which have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least about 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least about6 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C., and most preferably tostringent hybridization conditions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative positions that are the same. Also in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions that are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. For example, % identity values may begenerated by WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology266:460-480; http://blast.wustl/edu/blast/README.html). Further detailsregarding amino acid substitutions, which are considered conservativeunder such criteria, are provided below. Additional definitions areprovided throughout the subsections that follow.

Molecular Biology of and Uses for 36P6D5

As is further described in the Examples below, the 36P6D5 gene andprotein have been characterized using a number of analytical approaches.For example, analyses of nucleotide coding and amino acid sequences wereconducted in order to identify potentially related molecules, as well asrecognizable structural domains, topological features, and otherelements within the 36P6D5 mRNA and protein structure. Northern blotanalyses of 36P6D5 mRNA expression were conducted in order to establishthe range of normal and cancerous tissues expressing 36P6D5 message.

The 36P6D5 protein is predicted to be initially translated into a 235amino acid precursor containing a signal sequence which, duringpost-translational processing, is cleaved to yield a mature 211 or 212amino acid secreted protein (FIG. 1). The 36P6D5 protein has a predictedmolecular weight=25.9 and pI=8.97. 36P6D5 is predicted to be a membraneassociated protein, with the first 28 amino acids being intracellular.Based on the Signal Peptide algorithm, the intracellular portion of theprotein seems to be cleaved off between aa 29 and 30, releasing the restof the molecule as a soluble protein. The 36P6D5 gene is normallyexpressed predominantly in pancreas (FIG. 3), but is also expressed orover-expressed in several human cancers, including cancers of theprostate, breast, pancreas, colon, lung, bladder, kidney and ovary(FIGS. 4, 6 and 7). The 36P6D5 protein structure shows significanthomology to two previously described protein sequences, 2-19 proteinprecursor (Genbank P98173) (SEQ ID NO: 15) and an osteoblast proteindesignated GS3786 (Q92520) (SEQ ID NO: 16) (FIG. 2). 36P6D5 hassimilarities to a predicted osteoblast protein, NP-055703.1, with 36%identity over its entire sequence, with most of the 54% homologyoccurring between aa 74 and aa 235. It is also weakly similar to proteinGS3786. The fact that 36P6D5 has similarities to bone derived proteinsis not surprising as it was discovered using SSH comparing LAPC4AD(IT)and LAPC4AD(SQ) RNA. Given its homology to the osteoblast protein, it ispossible that the 36P6D5 protein may function as a secreted factor thatstimulates the proliferation of cancer cells (such as the 36P5D6 cancercells shown in FIGS. 4, 6 and 7) in bone.

As disclosed herein, 36P6D5 exhibits specific properties that areanalogous to those found in a family of genes whose polynucleotides,polypeptides and anti-polypeptide antibodies are used in well knowndiagnostic assays directed to examining conditions associated withdysregulated cell growth such as cancer, in particular prostate cancer(see e.g. both its highly specific pattern of tissue expression as wellas its overexpression in prostate cancers as described for example inExample 3). The best known member of this class is PSA, the archetypalmarker that has been used by medical practitioners for years to identifyand monitor the presence of prostate cancer (see e.g. Merrill et al., J.Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August;162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19):1635-1640(1999)). A variety of other diagnostic markers are also used inthis context including p53 and K-ras (see e.g. Tulchinsky et al., Int JMol Med 1999 July; 4(1):99-102 and Minimoto et al., Cancer Detect Prev2000; 24(1):1-12). Consequently, this disclosure of the 36P6D5polynucleotides and polypeptides (as well as the 36P6D5 polynucleotideprobes and anti-36P6D5 antibodies used to identify the presence of thesemolecules) and their properties allows skilled artisans to utilize thesemolecules in methods that are analogous to those used, for example, in avariety of diagnostic assays directed to examining conditions associatedwith cancer.

Typical embodiments of diagnostic methods which utilize the 36P6D5polynucleotides, polypeptides and antibodies described herein areanalogous to those methods from well established diagnostic assays whichemploy PSA polynucleotides, polypeptides and antibodies. For example,just as PSA polynucleotides are used as probes (for example in Northernanalysis, see e.g. Sharief et al., Biochem. Mol. Biol. Int.33(3):567-74(1994)) and primers (for example in PCR analysis, see e.g.Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe thepresence and/or the level of PSA mRNAs in methods of monitoring PSAoverexpression or the metastasis of prostate cancers, the 36P6D5polynucleotides described herein can be utilized in the same way todetect 36P6D5 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods ofmonitoring PSA protein overexpression (see e.g. Stephan et al., Urology55(4):560-3 (2000)) or the metastasis of prostate cells (see e.g. Alanenet al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 36P6D5polypeptides described herein can be utilized to generate antibodies foruse in detecting 36P6D5 overexpression or the metastasis of prostatecells and cells of other cancers expressing this gene. Specifically,because metastases involves the movement of cancer cells from an organof origin (such as the bladder, kidney or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 36P6D5polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis, for example, when a biological sample from tissue that doesnot normally contain 36P6D5 expressing cells (lymph node) is found tocontain 36P6D5 expressing cells. Alternatively 36P6D5 polynucleotidesand/or polypeptides can be used to provide evidence of cancer, forexample, when a cells in biological sample that do not normally express36P6D5 or express 36P6D5 at a different level (such as kidney, bladder,lung and prostate cells etc.) are found to express 36P6D5 or have anincreased expression of 36P6D5. In such assays, artisans may furtherwish to generate supplementary evidence of metastasis by testing thebiological sample for the presence of a second tissue restricted marker(in addition to 36P6D5) such as PSA, PSCA etc. (see e.g. Alanen et al.,Pathol. Res. Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring thismolecule, 36P6D5 polynucleotide fragments and polynucleotide variantscan also be used in an analogous manner. In particular, typical PSApolynucleotides used in methods of monitoring this molecule are probesor primers which consist of fragments of the PSA cDNA sequence.Illustrating this, primers used to PCR amplify a PSA polynucleotide mustinclude less than the whole PSA sequence to function in the polymerasechain reaction. In the context of such PCR reactions, skilled artisansgenerally create a variety of different polynucleotide fragments thatcan be used as primers in order to amplify different portions of apolynucleotide of interest or to optimize amplification reactions (seee.g. Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998);Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additionalillustration of the utility of such fragments is provided in Example 3,where a 36P6D5 polynucleotide fragment is used as a probe to show theoverexpression of 36P6D5 mRNAs in cancer cells. In addition, in order tofacilitate their use by medical practitioners, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see e.g. Sawai et al., Fetal Diagn.Ther. 1996 November-December; 11(6):407-13 and Current Protocols InMolecular Biology, Volume 2, Unit 2, Frederick M. Ausubul et al. eds.,1995)). Polynucleotide fragments and variants are typically useful inthis context as long as they have the common attribute or characteristicof being capable of binding to a target polynucleotide sequence (e.g.the 36P6D5 polynucleotide shown in SEQ ID NO: 1) under conditions ofrelatively high stringency.

Just as PSA polypeptide fragments and polypeptide variants are employedby skilled artisans for use in methods of monitoring this molecule,36P6D5 polypeptide fragments and polypeptide variants can also be usedin an analogous manner. In particular, typical PSA polypeptides used inmethods of monitoring this molecule are fragments of the PSA proteinwhich contain an epitope that can be recognized by an antibody whichwill specifically bind to the PSA protein. This practice of usingpolypeptide fragments or polypeptide variants used to generateantibodies (such as anti-PSA antibodies) is typical in the art with awide variety of systems such as fusion proteins being used bypractitioners (see e.g. Current Protocols In Molecular Biology, Volume2, Unit 16, Frederick M. Ausubul et al. eds., 1995). In this context,each of the variety of epitopes in a protein of interest functions toprovide the architecture upon which the antibody is generated.Typically, skilled artisans generally create a variety of differentpolypeptide fragments that can be used in order to generate antibodiesspecific for different portions of a polypeptide of interest (see e.g.U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 36P6D5biological motifs discussed below. Polypeptide fragments and variantsare typically useful in this context as long as they have the commonattribute or characteristic of having an epitope capable of generatingan antibody specific for a target polypeptide sequence (e.g. the 36P6D5polypeptide shown in SEQ ID NO: 2).

As shown herein, the 36P6D5 polynucleotides and polypeptides (as well asthe 36P6D5 polynucleotide probes and anti-36P6D5 antibodies used toidentify the presence of these molecules) exhibit specific propertiesthat make them useful in diagnosing cancers of the prostate. Thedescribed diagnostic assays that measures the presence of 36P6D5 geneproducts, in order to evaluate the presence or onset of the particulardisease conditions described herein such as prostate cancer areparticularly useful in identifying potential candidates for preventivemeasures or further monitoring, as has been done so successfully withPSA. Moreover, these materials satisfy a need in the art for moleculeshaving similar characteristics to PSA in situations where, for example,a definite diagnosis of metastasis of prostatic origin cannot be made onthe basis of a testing for PSA alone (see e.g. Alanen et al., Pathol.Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as36P6D5 polynucleotides and polypeptides (as well as the 36P6D5polynucleotide probes and anti-36P6D5 antibodies used to identify thepresence of these molecules) must be employed to confirm metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 36P6D5polynucleotides disclosed herein have a number of other specificutilities such as their use in the identification of oncogeneticassociated chromosomal abnormalities in 21q22.2-22.3. Moreover, inaddition to their use in diagnostic assays, the 36P6D5 polypeptides andpolynucleotides disclosed herein have other utilities such as their usein the forensic analysis of tissues of unknown origin (see e.g. TakahamaK Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).

As discussed in detail below, 36P6D5 function can be assessed inmammalian cells using a variety of techniques that are well known in theart. For mammalian expression, 36P6D5 can be cloned into severalvectors, including pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 36P6D5 can be expressed in several cell lines,including PC-3, NIH 3T3, LNCaP and 293T. Expression of 36P6D5 can bemonitored using northern blot analysis. The mammalian cell linesexpressing 36P6D5 can be tested in several in vitro and in vivo assays,including cell proliferation in tissue culture, activation of apoptoticsignals, tumor formation in SCID mice, and in vitro invasion using amembrane invasion culture system (MICS) (Welch et al., Int. J. Cancer43: 449-457). The 36P6D5 cell phenotype can be compared to the phenotypeof cells that lack expression of 36P6D5.

36P6D5 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 36P6D5 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 36P6D5 protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a 36P6D5 gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides that hybridize to a 36P6D5 gene,mRNA, or to a 36P6D5 encoding polynucleotide (collectively, “36P6D5polynucleotides”). As used herein, the 36P6D5 gene and protein is meantto include the 36P6D5 genes and proteins specifically described hereinand the genes and proteins corresponding to other 36P6D5 proteins andstructurally similar variants of the foregoing. Such other 36P6D5proteins and variants will generally have coding sequences that arehighly homologous to the 36P6D5 coding sequence, and preferably willshare at least about 50% amino acid identity and at least about 60%amino acid homology (using BLAST criteria), more preferably sharing 70%or greater homology (using BLAST criteria).

A 36P6D5 polynucleotide may comprise a polynucleotide having thenucleotide sequence of human 36P6D5 as shown in FIG. 1 (SEQ ID NO: 1),wherein T can also be U; a polynucleotide which encodes all or part ofthe 36P6D5 protein; a sequence complementary to the foregoing; or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide having the sequence as shown in FIG. 1 (SEQID NO: 1), from nucleotide residue number 59 through nucleotide residuenumber 763 or 766, wherein T can also be U. Another embodiment comprisesa polynucleotide having the sequence as shown in FIG. 1 (SEQ ID NO: 1),from nucleotide residue number 131 through nucleotide residue number 763or 766, wherein T can also be U. Another embodiment comprises apolynucleotide encoding a 36P6D5 polypeptide whose sequence is encodedby the cDNA contained in the plasmid as deposited on Apr. 9, 1999 withAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209, USA (ATCC) as Accession No. 207197. Another embodimentcomprises a polynucleotide which is capable of hybridizing understringent hybridization conditions to the human 36P6D5 cDNA shown inFIG. 1 (SEQ ID NO: 1).

Typical embodiments of the invention disclosed herein include 36P6D5polynucleotides containing specific portions of the 36P6D5 mRNA sequence(and those which are complementary to such sequences) such as those thatencode the protein and fragments thereof. For example, representativeembodiments of the invention disclosed herein include: polynucleotidesencoding about amino acid 1 to about amino acid 10 of the 36P6D5 proteinshown in SEQ ID NO: 2, polynucleotides encoding about amino acid 20 toabout amino acid 30 of the 36P6D5 protein shown in SEQ ID NO: 2,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 36P6D5 protein shown in SEQ ID NO: 2, polynucleotides encoding aboutamino acid 40 to about amino acid 50 of the 36P6D5 protein shown in SEQID NO: 2, polynucleotides encoding about amino acid 50 to about aminoacid 60 of the 36P6D5 protein shown in SEQ ID NO: 2, polynucleotidesencoding about amino acid 60 to about amino acid 70 of the 36P6D5protein shown in SEQ ID NO: 2, polynucleotides encoding about amino acid70 to about amino acid 80 of the 36P6D5 protein shown in SEQ ID NO: 2,polynucleotides encoding about amino acid 80 to about amino acid 90 ofthe 36P6D5 protein shown in SEQ ID NO: 2 and polynucleotides encodingabout amino acid 90 to about amino acid 100 of the 36P6D5 protein shownin SEQ ID NO: 2, etc. Following this scheme, polynucleotides encodingportions of the ammo acid sequence of amino acids 100-235 of the 36P6D5protein are typical embodiments of the invention. Polynucleotidesencoding larger portions of the 36P6D5 protein are also contemplated.For example polynucleotides encoding from about amino acid 1 (or 20 or30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the36P6D5 protein shown in SEQ ID NO: 2 may be generated by a variety oftechniques well known in the art.

Additional illustrative embodiments of 36P6D5 polynucleotides includeembodiments consisting of a polynucleotide having the sequence as shownin FIG. 1 (SEQ ID NO: 1) from about nucleotide residue number 1 throughabout nucleotide residue number 250, from about nucleotide residuenumber 250 through about nucleotide residue number 500 and from aboutnucleotide residue number 500 through about nucleotide residue number750 and from about nucleotide residue number 750 through aboutnucleotide residue number 913. These polynucleotide fragments caninclude any portion of the 36P6D5 sequence as shown in FIG. 1 (SEQ IDNO: 1), for example a polynucleotide having the 235 amino acid ORFwithin the polynucleotide sequence as shown in FIG. 1 (SEQ ID NO: 1),e.g. from about nucleotide residue number 59 through about nucleotideresidue number 763.

Additional illustrative embodiments of the invention disclosed hereininclude 36P6D5 polynucleotide fragments encoding one or more of thebiological motifs contained within the 36P6D5 protein sequence. Typicalpolynucleotide fragments of the invention include those that encode oneor more of the 36P6D5 N-glycosylation sites, casein kinase IIphosphorylation sites, the protein kinase c phosphorylation sites, theamino acid permeases signature or n-myristoylation sites as disclosed ingreater detail in the text discussing the 36P6D5 protein andpolypeptides below.

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. For example, because the human 36P6D5 gene mapsto chromosome 21q22.2-22.3, polynucleotides encoding different regionsof the 36P6D5 protein can be used to characterize cytogeneticabnormalities on chromosome 21, bands q22.2-22.3 that have beenidentified as being associated with various cancers. In particular, avariety of chromosomal abnormalities in 21q22.2-22.3 have beenidentified as frequent cytogenetic abnormalities in a number ofdifferent cancers (see, e.g., Babu et al., Cancer Genet Cytogenet. 1989March; 38(1):127-9 and Ho et al., Blood. 1996 Jun. 15; 87(12):5218-24).Consequently, polynucleotides encoding specific regions of the 36P6D5protein provide new tools that can be used to delineate with a greaterprecision than previously possible, the specific nature of thecytogenetic abnormalities in this region of chromosome 21 that maycontribute to the malignant phenotype. In this context, thesepolynucleotides satisfy a need in the art for expanding the sensitivityof chromosomal screening in order to identify more subtle and lesscommon chromosomal abnormalities (see, e.g., Evans et al., 1994, Am. J.Obstet. Gynecol. 171(4):1055-1057).

Alternatively, as 36P6D5 is shown to be highly expressed in prostatecancers (see e.g. FIG. 4), these polynucleotides may be used in methodsassessing the status of 36P6D5 gene products in normal versus canceroustissues. Typically, polynucleotides encoding specific regions of the36P6D5 protein may be used to assess the presence of perturbations (suchas deletions, insertions, point mutations etc.) in specific regions(such as regions containing the RNA binding sequences) of the 36P6D5gene products. Exemplary assays include both RT-PCR assays as well assingle-strand conformation polymorphism (SSCP) analysis (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8): 369-378), both of whichutilize polynucleotides encoding specific regions of a protein toexamine these regions within the protein.

Other specifically contemplated embodiments of the invention disclosedherein are genomic DNA, cDNAs, ribozymes, and antisense molecules, aswell as nucleic acid molecules based on an alternative backbone orincluding alternative bases, whether derived from natural sources orsynthesized. For example, antisense molecules can be RNAs or othermolecules, including peptide nucleic acids (PNAs) or non-nucleic acidmolecules such as phosphorothioate derivatives, that specifically bindDNA or RNA in a base pair-dependent manner. A skilled artisan canreadily obtain these classes of nucleic acid molecules using the 36P6D5polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,36P6D5. See for example, Jack Cohen, 1988, OLIGODEOXYNUCLEOTIDES,Antisense Inhibitors of Gene Expression, CRC Press; and Synthesis 1:1-5(1988). The 36P6D5 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention may beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See Iyer, R. P. et al, 1990, J. Org. Chem. 55:4693-4698; andIyer, R. P. et al., 1990, J. Am. Chem. Soc. 112:1253-1254, thedisclosures of which are fully incorporated by reference herein.Additional 36P6D5 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see e.g.Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6:169-175).

The 36P6D5 antisense oligonucleotides of the present invention typicallymay be RNA or DNA that is complementary to and stably hybridizes withthe first 100 N-terminal codons or last 100 C-terminal codons of the36P6D5 genomic sequence or the corresponding mRNA. While absolutecomplementarity is not required, high degrees of complementarity arepreferred. Use of an oligonucleotide complementary to this region allowsfor the selective hybridization to 36P6D5 mRNA and not to mRNAspecifying other regulatory subunits of protein kinase. Preferably, the36P6D5 antisense oligonucleotides of the present invention are a 15 to30-mer fragment of the antisense DNA molecule having a sequence thathybridizes to 36P6D5 mRNA. Optionally, 36P6D5 antisense oligonucleotideis a 30-mer oligonucleotide that is complementary to a region in thefirst 10 N-terminal codons and last 10 C-terminal codons of 36P6D5.Alternatively, the antisense molecules are modified to employ ribozymesin the inhibition of 36P6D5 expression (L. A. Couture & D. T.Stinchcomb, 1996, Trends Genet. 12: 510-515).

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a 36P6D5 polynucleotide in a sample and as ameans for detecting a cell expressing a 36P6D5 protein.

Examples of such probes include polynucleotides comprising all or partof the human 36P6D5 cDNA sequences shown in SEQ ID NO: 1. Examples ofprimer pairs capable of specifically amplifying 36P6D5 mRNAs are alsodescribed in the Examples that follow. As will be understood by theskilled artisan, a great many different primers and probes may beprepared based on the sequences provided herein and used effectively toamplify and/or detect a 36P6D5 mRNA.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than the 36P6D5 gene or that encodepolypeptides other than 36P6D5 gene product or fragments thereof. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated 36P6D5 polynucleotide.

The 36P6D5 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 36P6D5 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 36P6D5 polypeptides; as tools for modulatingor inhibiting the expression of the 36P6D5 gene(s) and/or translation ofthe 36P6D5 transcript(s); and as therapeutic agents.

Isolation of 36P6D5-Encoding Nucleic Acid Molecules

The 36P6D5 cDNA sequences described herein enable the isolation of otherpolynucleotides encoding 36P6D5 gene product(s), as well as theisolation of polynucleotides encoding 36P6D5 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 36P6D5 gene product. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a 36P6D5 gene are wellknown (See, e.g., Sambrook, J. et al., 1989, Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Press, New York; Ausubelet al., eds., 1995, Current Protocols in Molecular Biology, Wiley andSons). For example, lambda phage cloning methodologies may beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 36P6D5gene cDNAs may be identified by probing with a labeled 36P6D5 cDNA or afragment thereof. For example, in one embodiment, the 36P6D5 cDNA (SEQID NO: 1) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full length cDNAs corresponding to a 36P6D5gene. The 36P6D5 gene itself may be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 36P6D5 DNAprobes or primers.

Recombinant DNA Molecules and Host-vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 36P6D5 polynucleotide, including but not limited to phages, plasmids,phagemids, cosmids, YACs, BACs, as well as various viral and non-viralvectors well known in the art, and cells transformed or transfected withsuch recombinant DNA or RNA molecules. As used herein, a recombinant DNAor RNA molecule is a DNA or RNA molecule that has been subjected tomolecular manipulation in vitro. Methods for generating such moleculesare well known (see, e.g., Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 36P6D5 polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 or HighFive cell). Examplesof suitable mammalian cells include various prostate cancer cell linessuch as PrEC, LNCaP and TsuPr1, other transfectable or transducibleprostate cancer cell lines, as well as a number of mammalian cellsroutinely used for the expression of recombinant proteins (e.g., COS,CHO, 293, 293T cells). More particularly, a polynucleotide comprisingthe coding sequence of 36P6D5 may be used to generate 36P6D5 proteins orfragments thereof using any number of host-vector systems routinely usedand widely known in the art.

A wide range of host-vector systems suitable for the expression of36P6D5 proteins or fragments thereof are available (see, e.g., Sambrooket al., 1989, supra; Current Protocols in Molecular Biology, 1995,supra). Preferred vectors for mammalian expression include but are notlimited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vectorpSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expressionvectors, 36P6D5 may be preferably expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 36P6D5 protein or fragment thereof. Such host-vectorsystems may be employed to study the functional properties of 36P6D5 and36P6D5 mutations.

Recombinant human 36P6D5 protein may be produced by mammalian cellstransfected with a construct encoding 36P6D5. In an illustrativeembodiment described in the Examples, 293T cells can be transfected withan expression plasmid encoding 36P6D5, the 36P6D5 protein is expressedin the 293T cells, and the recombinant 36P6D5 protein can be isolatedusing standard purification methods (e.g., affinity purification usinganti-36P6D5 antibodies). In another embodiment, also described in theExamples herein, the 36P6D5 coding sequence is subcloned into theretroviral vector pSRαMSVtkneo and used to infect various mammalian celllines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish36P6D5 expressing cell lines. Various other expression systems wellknown in the art may also be employed. Expression constructs encoding aleader peptide joined in frame to the 36P6D5 coding sequence may be usedfor the generation of a secreted form of recombinant 36P6D5 protein.

Proteins encoded by the 36P6D5 genes, or by fragments thereof, will havea variety of uses, including but not limited to generating antibodiesand in methods for identifying ligands and other agents and cellularconstituents that bind to a 36P6D5 gene product. Antibodies raisedagainst a 36P6D5 protein (like 36P6D5 polynucleotides) or fragmentthereof may be useful in diagnostic and prognostic assays, and imagingmethodologies in the management of human cancers characterized byexpression of 36P6D5 protein, including but not limited to cancers ofthe kidney lung, colon, prostate, brain, bladder, pancreas, ovaries,lung, and breast (see e.g. FIGS. 4, 6 and 7). Such antibodies may beexpressed intracellularly and used in methods of treating patients withsuch cancers. Various immunological assays useful for the detection of36P6D5 proteins are contemplated, including but not limited to FACSanalysis, various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), immunocytochemical methods, and the like. Such antibodies maybe labeled and used as immunological imaging reagents capable ofdetecting 36P6D5 expressing cells (e.g., in radioscintigraphic imagingmethods). 36P6D5 proteins may also be particularly useful in generatingcancer vaccines, as further described below.

36P6D5 Polypeptides

Another aspect of the present invention provides 36P6D5 proteins andpolypeptide fragments thereof. The 36P6D5 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined below. Fusion proteins that combine partsof different 36P6D5 proteins or fragments thereof, as well as fusionproteins of a 36P6D5 protein and a heterologous polypeptide are alsoincluded. Such 36P6D5 proteins will be collectively referred to as the36P6D5 proteins, the proteins of the invention, or 36P6D5. As usedherein, the term “36P6D5 polypeptide” refers to a polypeptide fragmentor a 36P6D5 protein of at least 6 amino acids, preferably at least 15amino acids.

Specific embodiments of 36P6D5 proteins comprise a polypeptide havingthe amino acid sequence of human 36P6D5 as shown in SEQ ID NO: 2.Alternatively, embodiments of 36P6D5 proteins comprise variantpolypeptides having alterations in the amino add sequence of human36P6D5 as shown in SEQ ID NO: 2.

In general, naturally occurring allelic variants of human 36P6D5 willshare a high degree of structural identity and homology (e.g., 90% ormore identity). Typically, allelic variants of the 36P6D5 proteins willcontain conservative amino acid substitutions within the 36P6D5sequences described herein or will contain a substitution of an aminoacid from a corresponding position in a 36P6D5 homologue. One class of36P6D5 allelic variants will be proteins that share a high degree ofhomology with at least a small region of a particular 36P6D5 amino acidsequence, but will further contain a radical departure from thesequence, such as a non-conservative substitution, truncation, insertionor frame shift.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

Embodiments of the invention disclosed herein include a wide variety ofart accepted variants of 36P6D5 proteins such as polypeptides havingamino acid insertions, deletions and substitutions. 36P6D5 variants canbe made using methods known in the art such as site-directedmutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis (Carter et al., 1986, Nucl. Acids Res. 13:4331; Zoller etal., 1987, Nucl. Acids Res. 10:6487), cassette mutagenesis (Wells etal., 1985, Gene 34:315), restriction selection mutagenesis (Wells etal., 1986, Philos. Trans. R. Soc. London Ser. A, 317:415) or other knowntechniques can be performed on the cloned DNA to produce the 36P6D5variant DNA. Scanning ammo acid analysis can also be employed toidentify one or more amino acids along a contiguous sequence. Among thepreferred scanning amino acids are relatively small, neutral aminoacids. Such amino acids include alanine, glycine, serine, and cysteine.Alanine is typically a preferred scanning amino acid among this groupbecause it eliminates the side-chain beyond the beta-carbon and is lesslikely to alter the main-chain conformation of the variant. Alanine isalso typically preferred because it is the most common amino acid.Further, it is frequently found in both buried and exposed positions(Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, 1976, J.Mol. Biol., 150:1). If alanine substitution does not yield adequateamounts of variant, an isosteric amino acid can be used.

As defined herein, 36P6D5 variants have the distinguishing attribute ofhaving at least one epitope in common with a 36P6D5 protein having theamino acid sequence of SEQ ID NO: 2, such that an antibody thatspecifically binds to a 36P6D5 variant will also specifically bind tothe 36P6D5 protein having the amino acid sequence of SEQ ID NO: 2. Apolypeptide ceases to be a variant of the protein shown in SEQ ID NO: 2when it no longer contains an epitope capable of being recognized by anantibody that specifically binds to a 36P6D5 protein. Those skilled inthe art understand that antibodies that recognize proteins bind toepitopes of varying size, and a grouping of the order of about six aminoacids, contiguous or not, is regarded as a typical number of amino acidsin a minimal epitope. See e.g. Hebbes et al., Mol Immunol (1989)26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608. Asthere are approximately 20 amino acids that can be included at a givenposition within the minimal 6 amino acid epitope, an approximation ofthe odds of such an epitope occurring by chance are about 20⁶ or about 1in 64 million. Another specific class of 36P6D5 protein variants shares90% or more identity with the amino acid sequence of SEQ ID NO: 2.Another specific class of 36P6D5 protein variants comprises one or moreof the 36P6D5 biological motifs described below.

As discussed above, embodiments of the claimed invention includepolypeptides containing less than the 235 amino acid sequence of the36P6D5 protein shown in SEQ ID NO: 2 (and the polynucleotides encodingsuch polypeptides). For example, representative embodiments of theinvention disclosed herein include polypeptides consisting of aboutamino acid 1 to about amino acid 10 of the 36P6D5 protein shown in SEQID NO: 2, polypeptides consisting of about amino acid 20 to about aminoacid 30 of the 36P6D5 protein shown in SEQ ID NO: 2, polypeptidesconsisting of about amino acid 30 to about amino acid 40 of the 36P6D5protein shown in SEQ ID NO: 2, polypeptides consisting of about aminoacid 40 to about amino acid 50 of the 36P6D5 protein shown in SEQ ID NO:2, polypeptides consisting of about amino acid 50 to about amino acid 60of the 36P6D5 protein shown in SEQ ID NO: 2, polypeptides consisting ofabout amino acid 60 to about amino acid 70 of the 36P6D5 protein shownin SEQ ID NO: 2, polypeptides consisting of about amino acid 70 to aboutamino acid 80 of the 36P6D5 protein shown in SEQ ID NO: 2, polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the 36P6D5protein shown in SEQ ID NO: 2 and polypeptides consisting of about aminoacid 90 to about amino acid 100 of the 36P6D5 protein shown in SEQ IDNO: 2, etc. Following this scheme, polypeptides consisting of portionsof the amino acid sequence of amino acids 100-235 of the 36P6D5 proteinare typical embodiments of the invention. Polypeptides consisting oflarger portions of the 36P6D5 protein are also contemplated. For examplepolypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.)to about amino acid 20, (or 30, or 40 or 50 etc.) of the 36P6D5 proteinshown in SEQ ID NO: 2 may be generated by a variety of techniques wellknown in the art.

Additional illustrative embodiments of the invention disclosed hereininclude 36P6D5 polypeptides containing the amino acid residues of one ormore of the biological motifs contained within the 36P6D5 polypeptidesequence as shown in SEQ ID NO: 2. In one embodiment, typicalpolypeptides of the invention can contain one or more of the 36P6D5N-glycosylation sites such as NVTA at residues 120-123 and/or NHSD atresidues 208-211 (SEQ ID NO: 2). In another embodiment, typicalpolypeptides of the invention can contain one or more of the 36P6D5protein kinase C phosphorylation sites such as SIR at residues 43-45and/or STR at residues 160-162 (SEQ ID NO: 2). In another embodiment,typical polypeptides of the invention can contain one or more of the36P6D5 casein kinase II phosphorylation sites such as SIGE at residues46-49 and/or TYDD at residues 155-158 (SEQ ID NO: 2). In anotherembodiment, typical polypeptides of the invention can contain one ormore of the N-myristoylation sites such as GGGRSK at residues 81-86,GINIAI at residues 108-113 and/or GNVTAT at residues 119-124 (SEQ ID NO:2). In another embodiment, typical polypeptides of the invention cancontain the amino acid permease signatureAGGLLKVVFVVFASLCAWYSGYLLAELIPDAP at residues 5-36 (SEQ ID NO: 2). Inanother embodiment, typical polypeptides of the invention can containthe signal sequence shown in FIG. 1. In yet another embodiment, typicalpolypeptides of the invention can contain one or more immunogenicepitopes identified by a process described herein such as those shown inTable 1. Related embodiments of these inventions include polypeptidescontaining combinations of the different motifs discussed above withpreferable embodiments being those which contain no insertions,deletions or substitutions either within the motifs or the interveningsequences of these polypeptides. In addition, embodiments which includea number of either N-terminal and/or C-terminal amino acid residues oneither side of these motifs may be desirable (to, for example, include agreater portion of the polypeptide architecture in which the motif islocated). Typically the number of N-terminal and/or C-terminal aminoacid residues on either side of a motif is between about 5 to about 50amino acid residues.

Illustrative examples of such embodiments includes a polypeptide havingone or more motifs selected from the group consisting of SIR and/or SIGEand/or NHSD (SEQ ID NO: 2). Alternatively polypeptides having othercombinations of the biological motifs disclosed herein are alsocontemplated such as a polypeptide having SIR and any one of the otherbiological motifs such as SIGE or a polypeptide having TYDD and any oneof the other biological motifs such as GGGRSK etc. (SEQ ID NO: 2).

Polypeptides consisting of one or more of the 36P6D5 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 36P6D5 motifsdiscussed above are associated with growth dysregulation and because36P6D5 is overexpressed in cancers (FIGS. 4, 6 and 7). Casein kinase IIand protein kinase C for example are enzymes known to be associated withthe development of the malignant phenotype (see e.g. Chen et al., 1998,Lab Invest., 78(2):165-174; Gaiddon et al., 1995, Endocrinology136(10):4331-4338; Hall et al., 1996, Nucleic Acids Research24(6):1119-1126; Peterziel et al., 1999, Oncogene 18(46):6322-6329; andO'Brian, 1998, Oncol. Rep. 5(2): 305-309). Moreover, both glycosylationand myristoylation are protein modifications also associated with cancerand cancer progression (see e.g. Dennis et al., 1999, Biochim. Biophys.Acta 1473(1):21-34; Raju et al., 1997, Exp. Cell Res. 235(1):145-154).

The polypeptides of the preceding paragraphs have a number of differentspecific uses. As 36P6D5 is shown to be expressed in a variety ofcancers including kidney, prostate, bladder, ovarian, breast, pancreas,colon and lung cancer cell lines and/or patient samples (see e.g. FIGS.4, 6 and 7), these polypeptides may be used in methods assessing thestatus of 36P6D5 gene products in normal versus cancerous tissues andelucidating the malignant phenotype. Typically, polypeptides encodingspecific regions of the 36P6D5 protein may be used to assess thepresence of perturbations (such as deletions, insertions, pointmutations etc.) in specific regions of the 36P6D5 gene products (such asregions containing the RNA binding motifs). Exemplary assays can utilizeantibodies targeting a 36P6D5 polypeptide containing the amino acidresidues of one or more of the biological motifs contained within the36P6D5 polypeptide sequence in order to evaluate the characteristics ofthis region in normal versus cancerous tissues. Alternatively, 36P6D5polypeptides containing the amino acid residues of one or more of thebiological motifs contained within the 36P6D5 polypeptide sequence canbe used to screen for factors that interact with that region of 36P6D5.

As discussed above, redundancy in the genetic code permits variation in36P6D5 gene sequences. In particular, one skilled in the art willrecognize specific codon preferences by a specific host species and canadapt the disclosed sequence as preferred for a desired host. Forexample, preferred codon sequences typically have rare codons (i.e.,codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific organism may be calculated, forexample, by utilizing codon usage tables available on the Internet atthe following address: http://www.dna.affrc.go.jp/˜nakamura/codon.html.Nucleotide sequences that have been optimized for a particular hostspecies by replacing any codons having a usage frequency of less thanabout 20% are referred to herein as “codon optimized sequences.”

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that may be deleterious to gene expression. The GC content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. Where possible, the sequence may also be modified to avoidpredicted hairpin secondary mRNA structures. Other useful modificationsinclude the addition of a translational initiation consensus sequence atthe start of the open reading frame, as described in Kozak, 1989, Mol.Cell Biol., 9:5073-5080. Nucleotide sequences that have been optimizedfor expression in a given host species by elimination of spuriouspolyadenylation sequences, elimination of exon/intron splicing signals,elimination of transposon-like repeats and/or optimization of GC contentin addition to codon optimization are referred to herein as an“expression enhanced sequence.”

36P6D5 proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the 36P6D5 proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated 36P6D5 protein. A purified 36P6D5 proteinmolecule will be substantially free of other proteins or molecules thatimpair the binding of 36P6D5 to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a 36P6D5 protein include a purified 36P6D5 protein and afunctional, soluble 36P6D5 protein. In one form, such functional,soluble 36P6D5 proteins or fragments thereof retain the ability to bindantibody or other ligand.

The invention also provides 36P6D5 polypeptides comprising biologicallyactive fragments of the 36P6D5 amino acid sequence, such as apolypeptide corresponding to part of the amino acid sequence for 36P6D5as shown in SEQ ID NO: 2. Such polypeptides of the invention exhibitproperties of the 36P6D5 protein, such as the ability to elicit thegeneration of antibodies that specifically bind an epitope associatedwith the 36P6D5 protein.

36P6D5 polypeptides can be generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the artbased on the amino acid sequences of the human 36P6D5 proteins disclosedherein. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a polypeptide fragment of a 36P6D5protein. In this regard, the 36P6D5-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of36P6D5 proteins. 36P6D5 polypeptides are particularly useful ingenerating and characterizing domain specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of a36P6D5 protein), in identifying agents or cellular factors that bind to36P6D5 or a particular structural domain thereof and in varioustherapeutic contexts, including but not limited to cancer vaccines.

36P6D5 polypeptides containing particularly interesting structures canbe predicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-36P6D5 antibodies or in identifying cellular factors thatbind to 36P6D5.

In an embodiment described in the examples that follow, 36P6D5 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 36P6D5 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 36P6D5 protein intransfected cells. The secreted HIS-tagged 36P6D5 in the culture mediamay be purified using a nickel column using standard techniques.

Modifications of 36P6D5 such as covalent modifications are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of a 36P6D5 polypeptidewith an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues of the 36P6D5.Another type of covalent modification of the 36P6D5 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence 36P6D5(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequence36P6D5. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present. Anothertype of covalent modification of 36P6D5 comprises linking the 36P6D5polypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The 36P6D5 of the present invention may also be modified in a way toform a chimeric molecule comprising 36P6D5 fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of the 36P6D5 with apolyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the 36P6D5. In analternative embodiment, the chimeric molecule may comprise a fusion ofthe 36P6D5 with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a 36P6D5 polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

36P6D5 Antibodies

The term “antibody” is used in the broadest sense and specificallycovers single anti-36P6D5 monoclonal antibodies (including agonist,antagonist and neutralizing antibodies) and anti-36P6D5 antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” (mAb) as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the antibodiescomprising the individual population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

Another aspect of the invention provides antibodies that bind to 36P6D5proteins and polypeptides. The most preferred antibodies willspecifically bind to a 36P6D5 protein and will not bind (or will bindweakly) to non-36P6D5 proteins and polypeptides. Anti-36P6D5 antibodiesthat are particularly contemplated include monoclonal and polyclonalantibodies as well as fragments containing the antigen binding domainand/or one or more complementarity determining regions of theseantibodies. As used herein, an antibody fragment is defined as at leasta portion of the variable region of the immunoglobulin molecule thatbinds to its target, i.e., the antigen binding region.

36P6D5 antibodies of the invention may be particularly useful inprostate cancer diagnostic and prognostic assays, and imagingmethodologies. Intracellularly expressed antibodies (e.g., single chainantibodies) may be therapeutically useful in treating cancers in whichthe expression of 36P6D5 is involved, such as for example advanced andmetastatic prostate cancers. Such antibodies may be useful in thetreatment, diagnosis, and/or prognosis of other cancers, to the extent36P6D5 is also expressed or overexpressed in other types of cancers suchas prostate, kidney, bladder, ovarian, breast, pancreas, colon and lungcancers.

36P6D5 antibodies may also be used therapeutically by, for example,modulating or inhibiting the biological activity of a 36P6D5 protein ortargeting and destroying cancer cells expressing a 36P6D5 protein or36P6D5 binding partner. Because 36P6D5 is a secreted protein, antibodiesmay be therapeutically useful for blocking 36P6D5's ability to bind toits receptor or interact with other proteins through which it exerts itsbiological activity.

The invention also provides various immunological assays useful for thebinding to, detection and quantification of 36P6D5 and mutant 36P6D5proteins and polypeptides. Such methods and assays generally compriseone or more 36P6D5 antibodies capable of recognizing and binding a36P6D5 or mutant 36P6D5 protein, as appropriate, and may be performedwithin various immunological assay formats well known in the art,including but not limited to various types of radioimmunoassays,enzyme-linked immunosorbent assays (ELISA), enzyme-linkedimmunofluorescent assays (ELIFA), and the like. In addition,immunological imaging methods capable of detecting prostate cancer andother cancers expressing 36P6D5 are also provided by the invention,including but limited to radioscintigraphic imaging methods usinglabeled 36P6D5 antibodies. Such assays may be clinically useful in thedetection, monitoring, and prognosis of 36P6D5 expressing cancers suchas prostate, breast, pancreas, colon and ovarian cancer cell lines.

36P6D5 antibodies may also be used in methods for purifying 36P6D5 andmutant 36P6D5 proteins and polypeptides and for isolating 36P6D5homologues and related molecules. For example, in one embodiment, themethod of purifying a 36P6D5 protein comprises incubating a 36P6D5antibody, which has been coupled to a solid matrix, with a lysate orother solution containing 36P6D5 under conditions that permit the 36P6D5antibody to bind to 36P6D5; washing the solid matrix to eliminateimpurities; and eluting the 36P6D5 from the coupled antibody. Other usesof the 36P6D5 antibodies of the invention include generatinganti-idiotypic antibodies that mimic the 36P6D5 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a 36P6D5 protein, peptide, or fragment, in isolatedor immunoconjugated form (Harlow, and Lane, eds., 1988, Antibodies: ALaboratory Manual, CSH Press; Harlow, 1989, Antibodies, Cold SpringHarbor Press, NY). In addition, fusion proteins of 36P6D5 may also beused, such as a 36P6D5 GST-fusion protein. In a particular embodiment, aGST fusion protein comprising all or most of the open reading frameamino acid sequence of SEQ ID NO: 2 may be produced and used as animmunogen to generate appropriate antibodies. In another embodiment, a36P6D5 peptide may be synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art may beused (with or without purified 36P6D5 protein or 36P6D5 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

The amino acid sequence of the 36P6D5 as shown in SEQ ID NO: 2 may beused to select specific regions of the 36P6D5 protein for generatingantibodies. For example, hydrophobicity and hydrophilicity analyses ofthe 36P6D5 amino acid sequence may be used to identify hydrophilicregions in the 36P6D5 structure. Regions of the 36P6D5 protein that showimmunogenic structure, as well as other regions and domains, can readilybe identified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis.

Illustrating this, the binding of peptides from 36P6D5 proteins to thehuman MHC class I molecule HLA-A2 are predicted and shown in Table 1below. Specifically, the complete amino acid sequences of 36P6D5proteins was entered into the HLA Peptide Motif Search algorithm foundin the Bioinformatics and Molecular Analysis Section (BIMAS) Web site(http://bimas.dcrt.nih.gov/). The HLA Peptide Motif Search algorithm wasdeveloped by Dr. Ken Parker based on binding of specific peptidesequences in the groove of HLA Class I molecules and specifically HLA-A2(see e.g. Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parkeret al., J. Immunol. 152:163-75 (1994)). This algorithm allows locationand ranking of 8-mer, 9-mer, and 10-mer peptides from a complete proteinsequence for predicted binding to HLA-A2 as well as other HLA Class Imolecules. Most HLA-A2 binding peptides are 9-mers favorably containinga leucine (L) at position 2 and a valine (V) or leucine (L) at position9 (Parker et al., J. Immunol. 149:3580-7 (1992)). The results of 36P6D5predicted binding peptides are shown in Table 1 below. In Table 1, thetop 10 ranking candidates for each family member are shown along withtheir location, the amino acid sequence of each specific peptide, and anestimated binding score. The binding score corresponds to the estimatedhalf-time of dissociation of complexes containing the peptide at 37° C.at pH 6.5. Peptides with the highest binding score are predicted to bethe most tightly bound to HLA Class I on the cell surface and thusrepresent the best immunogenic targets for T-cell recognition. Actualbinding of peptides to HLA-A2 can be evaluated by stabilization ofHLA-A2 expression on the antigen-processing defective cell line T2 (seee.g. Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate36:129-38 (1998)). Immunogenicity of specific peptides can be evaluatedin vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in thepresence of dendritic cells.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a 36P6D5 immunogen is conducted generallyby injection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

36P6D5 monoclonal antibodies are preferred and may be produced byvarious means well known in the art. For example, immortalized celllines that secrete a desired monoclonal antibody may be prepared usingthe standard hybridoma technology of Kohler and Milstein ormodifications that immortalize producing B cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the 36P6D5 protein or a36P6D5 fragment. When the appropriate immortalized cell culturesecreting the desired antibody is identified, the cells may be expandedand antibodies produced either from in vitro cultures or from ascitesfluid.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the 36P6D5 protein can also be produced in thecontext of chimeric or CDR grafted antibodies of multiple speciesorigin. Humanized or human 36P6D5 antibodies may also be produced andare preferred for use in therapeutic contexts. Methods for humanizingmurine and other non-human antibodies by substituting one or more of thenon-human antibody CDRs for corresponding human antibody sequences arewell known (see for example, Jones et al., 1986, Nature 321:522-525;Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988,Science 239:1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad.Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151:2296. Methodsfor producing fully human monoclonal antibodies include phage displayand transgenic methods (for review, see Vaughan et al., 1998, NatureBiotechnology 16:535-539).

Fully human 36P6D5 monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In: Clark,M., ed., 1993, Protein Engineering of Antibody Molecules forProphylactic and Therapeutic Applications in Man, Nottingham Academic,pp 45-64; Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human 36P6D5 monoclonal antibodies mayalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4):607-614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 36P6D5 antibodies with a 36P6D5 protein may be establishedby a number of well known means, including western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,36P6D5 proteins, peptides, 36P6D5-expressing cells or extracts thereof.

A 36P6D5 antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. A second molecule forconjugation to the 36P6D5 antibody can be selected in accordance withthe intended use. For example, for therapeutic use, the second moleculecan be a toxin or therapeutic agent. Further, bi-specific antibodiesspecific for two or more 36P6D5 epitopes may be generated using methodsgenerally known in the art. Homodimeric antibodies may also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., 1993,Cancer Res. 53: 2560-2565).

36P6D5 Transgenic Animals

Nucleic acids that encode 36P6D5 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA that is integrated into the genomeof a cell from which a transgenic animal develops. In one embodiment,cDNA encoding 36P6D5 can be used to clone genomic DNA encoding 36P6D5 inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain cells that express DNA encoding36P6D5. Methods for generating transgenic animals, particularly animalssuch as mice or rats, have become conventional in the art and aredescribed, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for 36P6D5 transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding 36P6D5 introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding 36P6D5. Such animals canbe used as tester animals for reagents thought to confer protectionfrom, for example, pathological conditions associated with itsoverexpression. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of 36P6D5 can be used to construct a36P6D5 “knock out” animal that has a defective or altered gene encoding36P6D5 as a result of homologous recombination between the endogenousgene encoding 36P6D5 and altered genomic DNA encoding 36P6D5 introducedinto an embryonic cell of the animal. For example, cDNA encoding 36P6D5can be used to clone genomic DNA encoding 36P6D5 in accordance withestablished techniques. A portion of the genomic DNA encoding 36P6D5 canbe deleted or replaced with another gene, such as a gene encoding aselectable marker that can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector (see e.g., Thomas and Capecchi, 1987, Cell51:503) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see e.g., Li et al.,1992, Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras (see e.g., Bradley, in Robertson, ed., 1987, Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, (IRL, Oxford), pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the 36P6D5 polypeptide.

Methods for the Detection of 36P6D5

Another aspect of the present invention relates to methods for detecting36P6D5 polynucleotides and 36P6D5 proteins and variants thereof, as wellas methods for identifying a cell that expresses 36P6D5. The expressionprofile of 36P6D5 makes it a potential diagnostic marker for localand/or metastasized disease. Northern blot analysis suggests thatdifferent tissues express different isoforms of 36P6D5. The 36P6D5isoforms in prostate cancer appear to be different from the isoformexpressed in normal prostate. In this context, the status of 36P6D5 geneproducts may provide information useful for predicting a variety offactors including susceptibility to advanced stage disease, rate ofprogression, and/or tumor aggressiveness. As discussed in detail below,the status of 36P6D5 gene products in patient samples may be analyzed bya variety protocols that are well known in the art includingimmunohistochemical analysis, the variety of Northern blottingtechniques including in situ hybridization, RT-PCR analysis (for exampleon laser capture micro-dissected samples), western blot analysis andtissue array analysis.

More particularly, the invention provides assays for the detection of36P6D5 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 36P6D5 polynucleotides include, for example, a 36P6D5gene or fragments thereof, 36P6D5 mRNA, alternative splice variant36P6D5 mRNAs, and recombinant DNA or RNA molecules containing a 36P6D5polynucleotide. A number of methods for amplifying and/or detecting thepresence of 36P6D5 polynucleotides are well known in the art and may beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 36P6D5 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a36P6D5 polynucleotides as sense and antisense primers to amplifying36P6D5 cDNAs therein; and detecting the presence of the amplified 36P6D5cDNA. Optionally, the sequence of the amplified 36P6D5 cDNA can bedetermined. In another embodiment, a method of detecting a 36P6D5 genein a biological sample comprises first isolating genomic DNA from thesample; amplifying the isolated genomic DNA using 36P6D5 polynucleotidesas sense and antisense primers to amplify the 36P6D5 gene therein; anddetecting the presence of the amplified 36P6D5 gene. Any number ofappropriate sense and antisense probe combinations may be designed fromthe nucleotide sequences provided for the 36P6D5 (SEQ ID NO: 1) and usedfor this purpose.

The invention also provides assays for detecting the presence of a36P6D5 protein in a tissue of other biological sample such as serum,bone, prostate, and other tissues, urine, cell preparations, and thelike. Methods for detecting a 36P6D5 protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,Western Blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, in one embodiment, a method of detecting the presenceof a 36P6D5 protein in a biological sample comprises first contactingthe sample with a 36P6D5 antibody, a 36P6D5-reactive fragment thereof,or a recombinant protein containing an antigen binding region of a36P6D5 antibody; and then detecting the binding of 36P6D5 protein in thesample thereto.

Methods for identifying a cell that expresses 36P6D5 are also provided.In one embodiment, an assay for identifying a cell that expresses a36P6D5 gene comprises detecting the presence of 36P6D5 mRNA in the cell.Methods for the detection of particular mRNAs in cells are well knownand include, for example, hybridization assays using complementary DNAprobes (such as in situ hybridization using labeled 36P6D5 riboprobes,Northern blot and related techniques) and various nucleic acidamplification assays (such as RT-PCR using complementary primersspecific for 36P6D5, and other amplification type detection methods,such as, for example, branched DNA, SISBA, TMA and the like).Alternatively, an assay for identifying a cell that expresses a 36P6D5gene comprises detecting the presence of 36P6D5 protein in the cell orsecreted by the cell. Various methods for the detection of proteins arewell known in the art and may be employed for the detection of 36P6D5proteins and 36P6D5 expressing cells.

36P6D5 expression analysis may also be useful as a tool for identifyingand evaluating agents that modulate 36P6D5 gene expression. For example,36P6D5 expression is significantly upregulated in prostate cancer, andmay also be expressed in other cancers. Identification of a molecule orbiological agent that could inhibit 36P6D5 expression or over-expressionin cancer cells may be of therapeutic value. Such an agent may beidentified by using a screen that quantifies 36P6D5 expression byRT-PCR, nucleic acid hybridization or antibody binding.

Assays for Circulating and Excreted 36P6D5

Mature 36P6D5 protein has an N-terminal signal sequence in the cDNAencoded ORF. Because 36P6D5 is a secreted protein expressed in cancersof the prostate, kidney, bladder, breast, colon, ovary, pancreas, andpossibly other cancers, tumors expressing 36P6D5 would be expected tosecrete 36P6D5 into the vasculature, and/or excreted into urine orsemen, where the protein may be detected and quantified using assays andtechniques well known in the molecular diagnostic field. Detecting andquantifying the levels of circulating or excreted 36P6D5 is expected tohave a number of uses in the diagnosis, staging, and prognosis ofcancers expressing 36P6D5, including but not limited to cancers of theprostate, kidney, bladder, breast, colon, ovary and pancreas. A numberof different technical approaches for the detection and quantificationof proteins in serum, urine or semen are well known in the art.

Because 36P6D5 is a secreted protein expressed in cancers of theprostate, kidney, bladder, breast, colon, ovary, pancreas, and possiblyother cancers, assays for detecting and quantifying 36P6D5 in blood orserum are expected to be useful for the detection, diagnosis, prognosis,and/or staging of a 36P6D5 expressing tumor in an individual. Forexample, 36P6D5 expression in normal tissues is found predominantly inpancreas (FIG. 3), with lower levels of expression detected in prostateand small intestine. However, high level expression is detected inxenografts derived from prostate cancer as well as cell lines derivedfrom cancers of the breast, colon, pancreas and ovary (FIG. 4).Accordingly, detection of serum 36P6D5 may provide an indication of thepresence of a prostate, breast, colon, ovarian or pancreatic tumor.Diagnosis of cancer may be made on the basis of this information and/orother information. In respect of prostate cancer, for example, suchother information may include serum PSA measurements, DRE and/orultrasonography. Further, the level of 36P6D5 detected in the serum mayprovide information useful in staging or prognosis. For example, assupported by the data presented in FIGS. 9 and 10, as cell populationsexpand and growth through the stroma and microvasculature, higher levelsof 36P6D5 are expected to be observed in serum. In this context, veryhigh levels of 36P6D5 protein in serum may suggest larger and/or moreaggressive tumors.

In addition, peripheral blood and bone marrow may be convenientlyassayed for the presence of 36P6D5 protein and/or 36P6D5 expressingcancer cells, including but not limited to prostate, bladder, colon,pancreatic, kidney and ovarian cancers, using RT-PCR to detect 36P6D5expression. The presence of RT-PCR amplifiable 36P6D5 mRNA provides anindication of the presence of the cancer. RT-PCR detection assays fortumor cells in peripheral blood are currently being evaluated for use inthe diagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195-2000; Heston etal., 1995, Clin. Chem. 41: 1687-1688). RT-PCR assays are well known inthe art.

In one embodiment, a capture ELISA is used to detect and quantify 36P6D5in serum, urine or semen. A capture ELISA for 36P6D5 comprises,generally, at least two monoclonal antibodies of different isotypes thatrecognize distinct epitopes of the 36P6D5 protein, or one anti-36P6D5monoclonal antibody and a specific polyclonal serum derived from adifferent species (e.g., rabbit, goat, sheep, hamster, etc.). In thisassay, one reagent serves as the capture (or coating) antibody and theother as the detection antibody. As shown in Table 2, clinical serumsamples from and pancreatic cancer patients, and normal male donors werescreened for 36P6D5 protein using a capture ELISA as described above andin FIG. 10. Supernatants from PC-3-36P6D5 and Du4475 cells, and fromPC-3-neo cells, served as positive and negative controls, respectivelyfor 36P6D5 protein detection. ND: not detected or below detectionsensitivity. 36P6D5 protein was detected in 7/10 colon cancer patientsand 6/10 pancreatic cancer patients, one of which had relatively highlevels (29.70 ng/ml), but only in 1/6 normal male donors. In a relatedembodiment, FACS analysis is used for the detection of cells expressing36P6D5 protein, for example those that have escaped the local site ofdisease and have migrated to other sites such as the lymphatic system.

As discussed in detail below, levels of 36P6D5 including 36P6D5 serumlevels may be used to provide an indication of the presence, extent andaggressiveness of a 36P6D5 expressing tumor. As noted, above 36P6D5shares a number of characteristics with PSA which is the most important,accurate, and clinically useful biochemical marker in the prostate. Anyprocess that disrupts the normal architecture of the prostate allowsdiffusion of PSA into the stroma and microvasculature. Consequently,clinically important increases in serum prostate-specific antigen levelsare seen with prostatic cancers. In particular, the greater number ofmalignant cells and the stromal disruption associated with canceraccount for the increased serum prostate-specific antigen level. In thiscontext, serum prostate-specific antigen levels correlate positivelywith clinical stage, tumor volume, histologic grade, and the presence ofcapsular perforation and seminal vesicle invasion. See e.g. Bostwick, D.G. Am. J. Clin. Pathol. 102 (4 Suppl 1): S31-S37 (1994).

Using PSA as an analogous molecule, is likely that because 36P6D5 isalso a secreted molecule that exhibits a restricted pattern of tissueexpression (including the prostate), the increasing load of malignantcells and the stromal disruption that occurs with cancer will make theserum 36P6D5 antigen levels correlate positively with one or moreclinically relevant factors such as clinical stage, tumor volume,histologic grade, and the presence of capsular perforation and seminalvesicle invasion. Serum 36P6D5 measurements over time would be expectedto provide further information, wherein an increase in 36P6D5 would beexpected to reflect progression and the rate of the increase would beexpected to correlate with aggressiveness. Similarly, a decline in serum36P6D5 would be expected to reflect a slower growing or regressingtumor. The identification of 36P6D5 in serum may be useful to detecttumor initiation and early stage disease. In patients who have undergonesurgery or therapy, serum 36P6D5 levels would be useful for monitoringtreatment response and potential recurrence.

Monitoring the Status of 36P6D5 and its Products

Assays that evaluate the status of the 36P6D5 gene and 36P6D5 geneproducts in an individual may provide information on the growth oroncogenic potential of a biological sample from this individual. Forexample, because 36P6D5 mRNA is so highly expressed in prostate cancersas compared to normal prostate tissue, assays that evaluate the relativelevels of 36P6D5 mRNA transcripts or proteins in a biological sample maybe used to diagnose a disease associated with 36P6D5 dysregulation suchas cancer and may provide prognostic information useful in definingappropriate therapeutic options.

Because 36P6D5 is expressed, for example, in various prostate cancerxenograft tissues and cancer cell lines, and cancer patient samples, theexpression status of 36P6D5 can provide information useful fordetermining information including the presence, stage and location ofdisplasic, precancerous and cancerous cells, predicting susceptibilityto various stages of disease, and/or for gauging tumor aggressiveness.Moreover, the expression profile makes it a potential imaging reagentfor metastasized disease. Consequently, an important aspect of theinvention is directed to the various molecular prognostic and diagnosticmethods for examining the status of 36P6D5 in biological samples such asthose from individuals suffering from, or suspected of suffering from apathology characterized by dysregulated cellular growth such as cancer.

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see e.g.Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant 36P6D5expression in prostate cancers) can allow the early detection of suchaberrant cellular physiology before a pathology such as cancer hasprogressed to a stage at which therapeutic options are more limited. Insuch examinations, the status of 36P6D5 in a biological sample ofinterest (such as one suspected of having dysregulated cell growth) canbe compared, for example, to the status of 36P6D5 in a correspondingnormal sample (e.g. a sample from that individual (or alternativelyanother individual) that is not effected by a pathology, for example onenot suspected of having dysregulated cell growth) with alterations inthe status of 36P6D5 in the biological sample of interest (as comparedto the normal sample) providing evidence of dysregulated cellulargrowth. In addition to using a biological sample that is not effected bya pathology as a normal sample, one can also use a predeterminednormative value such as a predetermined normal level of mRNA expression(see e.g. Grever et al., J. Comp. Neurol. 1996 Dec. 9; 376(2):306-14 andU.S. Pat. No. 5,837,501) to compare 36P6D5 in normal versus suspectsamples.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.As specifically described herein, the status of 36P6D5 can be evaluatedby a number of parameters known in the art. Typically an alteration inthe status of 36P6D5 comprises a change in the location of 36P6D5expressing cells and/or an increase in 36P6D5 mRNA and/or proteinexpression.

Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 36P6D5 expressing cells) as well as the, level, andbiological activity of expressed gene products (such as 36P6D5 mRNApolynucleotides and polypeptides). Alterations in the status of 36P6D5can be evaluated by a wide variety of methodologies well known in theart, typically those discussed below. Typically an alteration in thestatus of 36P6D5 comprises a change in the location of 36P6D5 and/or36P6D5 expressing cells and/or an increase in 36P6D5 mRNA and/or proteinexpression.

As discussed in detail herein, in order to identify a condition orphenomenon associated with dysregulated cell growth, the status of36P6D5 in a biological sample may be evaluated by a number of methodsutilized by skilled artisans including, but not limited to genomicSouthern analysis (to examine, for example perturbations in the 36P6D5gene), northerns and/or PCR analysis of 36P6D5 mRNA (to examine, forexample alterations in the polynucleotide sequences or expression levelsof 36P6D5 mRNAs), and western and/or immunohistochemical analysis (toexamine, for example alterations in polypeptide sequences, alterationsin polypeptide localization within a sample, alterations in expressionlevels of 36P6D5 proteins and/or associations of 36P6D5 proteins withpolypeptide binding partners). Detectable 36P6D5 polynucleotidesinclude, for example, a 36P6D5 gene or fragments thereof, 36P6D5 mRNA,alternative splice variants 36P6D5 mRNAs, and recombinant DNA or RNAmolecules containing a 36P6D5 polynucleotide.

The expression profile of 36P6D5 makes it a potential diagnostic markerfor local and/or metastasized disease. In particular, the status of36P6D5 may provide information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 36P6D5 status anddiagnosing cancers that express 36P6D5, such as cancers of the prostate,bladder, bladder, kidney, ovaries, breast, pancreas, colon and lung.36P6D5 status in patient samples may be analyzed by a number of meanswell known in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, western blot analysis of clinical samples andcell lines, and tissue array analysis. Typical protocols for evaluatingthe status of the 36P6D5 gene and gene products can be found, forexample in Ausubul et al. eds., 1995, Current Protocols In MolecularBiology, Units 2 [Northern Blotting], 4 [Southern Blotting], 15[Immunoblotting] and 18 [PCR Analysis].

As described above, the status of 36P6D5 in a biological sample can beexamined by a number of well known procedures in the art. For example,the status of 36P6D5 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 36P6D5 expressing cells (e.g. those that express36P6D5 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth for example, when 36P6D5 expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node). Such alterations in the status of 36P6D5in a biological sample are often associated with dysregulated cellulargrowth. Specifically, one indicator of dysregulated cellular growth isthe metastases of cancer cells from an organ of origin (such as thebladder, kidney or prostate gland) to a different area of the body (suchas a lymph node). In this context, evidence of dysregulated cellulargrowth is important for example because occult lymph node metastases canbe detected in a substantial proportion of patients with prostatecancer, and such metastases are associated with known predictors ofdisease progression (see e.g. J Urol 1995 August; 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 36P6D5 geneproducts by determining the status of 36P6D5 gene products expressed bycells in a test tissue sample from an individual suspected of having adisease associated with dysregulated cell growth (such as hyperplasia orcancer) and then comparing the status so determined to the status of36P6D5 gene products in a corresponding normal sample, the presence ofaberrant 36P6D5 gene products in the test sample relative to the normalsample providing an indication of the presence of dysregulated cellgrowth within the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 36P6D5 mRNA or protein expression in a test cellor tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 36P6D5 mRNA may, for example, beevaluated in tissue samples including but not limited to prostate,kidney, bladder, ovarian, breast, pancreas, colon and lung issues (seee.g. FIGS. 4, 6 and 7). The presence of significant 36P6D5 expression inany of these tissues may be useful to indicate the emergence, presenceand/or severity of these cancers, since the corresponding normal tissuesdo not express 36P6D5 mRNA or express it at lower levels.

In a related embodiment, 36P6D5 status may be determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodor assay would comprise determining the level of 36P6D5 proteinexpressed by cells in a test tissue sample and comparing the level sodetermined to the level of 36P6D5 expressed in a corresponding normalsample. In one embodiment, the presence of 36P6D5 protein is evaluated,for example, using immunohistochemical methods. 36P6D5 antibodies orbinding partners capable of detecting 36P6D5 protein expression may beused in a variety of assay formats well known in the art for thispurpose.

In other related embodiments, one can evaluate the integrity 36P6D5nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like. Such embodiments areuseful because perturbations in the nucleotide and amino acid sequencesare observed in a large number of proteins associated with a growthdysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan.Pathol. 26(8):369-378). In this context, a wide variety of assays forobserving perturbations in nucleotide and amino acid sequences are wellknown in the art. For example, the size and structure of nucleic acid oramino acid sequences of 36P6D5 gene products may be observed by theNorthern, Southern, Western, PCR and DNA sequencing protocols discussedherein. In addition, other methods for observing perturbations innucleotide and amino acid sequences such as single strand conformationpolymorphism analysis are well known in the art (see, e.g., U.S. Pat.Nos. 5,382,510 and 5,952,170).

In another embodiment, one can examine the methylation status of the36P6D5 gene in a biological sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells and can result in alteredexpression of various genes. For example, promoter hypermethylation ofthe pi-class glutathione S-transferase (a protein expressed in normalprostate but not expressed in >90% of prostate carcinomas) appears topermanently silence transcription of this gene and is the mostfrequently detected genomic alteration in prostate carcinomas (De Marzoet al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, thisalteration is present in at least 70% of cases of high-grade prostaticintraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol.Biomarkers Prev., 1998, 7:531-536). In another example, expression ofthe LAGE-I tumor specific gene (which is not expressed in normalprostate but is expressed in 25-50% of prostate cancers) is induced bydeoxy-azacytidine in lymphoblastoid cells, suggesting that tumoralexpression is due to demethylation (Lethe et al., Int. J. Cancer 76(6):903-908 (1998)). In this context, a variety of assays for examiningmethylation status, of a gene are well known in the art. For example,one can utilize in Southern hybridization approachesmethylation-sensitive restriction enzymes which can not cleave sequencesthat contain methylated CpG sites in order to assess the overallmethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Units 12, Frederick M. Ausubulet al. eds., 1995.

Gene amplification provides an additional method of assessing the statusof 36P6D5, a locus that maps to 21q22.2-22.3, a region shown to beperturbed in a variety of cancers. Gene amplification may be measured ina sample directly, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, 1980,Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis),or in situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

In addition to the tissues discussed above, biopsied tissue orperipheral blood or bone marrow may be conveniently assayed for thepresence of cancer cells, including but not limited to prostate, kidney,bladder, ovarian, breast, pancreas, colon and lung cancers using forexample, Northern, dot blot or RT-PCR analysis to detect 36P6D5expression (see e.g. FIGS. 4, 6 and 7). The presence of RT-PCRamplifiable 36P6D5 mRNA provides an indication of the presence of thecancer. RT-PCR detection assays for tumor cells in peripheral blood arecurrently being evaluated for use in the diagnosis and management of anumber of human solid tumors. In the prostate cancer field, theseinclude RT-PCR assays for the detection of cells expressing PSA and PSM(Verkaik et al, 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J.Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem.41:1687-1688). RT-PCR assays are well known in the art.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detecting36P6D5 mRNA or 36P6D5 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 36P6D5 mRNAexpression present is proportional to the degree of susceptibility. In aspecific embodiment, the presence of 36P6D5 in prostate tissue isexamined, with the presence of 36P6D5 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). In another specific embodiment, thepresence of 36P6D5 in tissue is examined, with the presence of 36P6D5 inthe sample providing an indication of cancer susceptibility (or theemergence or existence of a tumor). In a closely related embodiment, onecan evaluate the integrity 36P6D5 nucleotide and amino acid sequences ina biological sample in order to identify perturbations in the structureof these molecules such as insertions, deletions, substitutions and thelike, with the presence of one or more perturbations in 36P6D5 geneproducts in the sample providing an indication of cancer susceptibility(or the emergence or existence of a tumor).

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of 36P6D5 mRNAor 36P6D5 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 36P6D5 mRNA or 36P6D5 proteinexpressed in a corresponding normal tissue taken from the sameindividual or a normal tissue reference sample, wherein the degree of36P6D5 mRNA or 36P6D5 protein expression in the tumor sample relative tothe normal sample indicates the degree of aggressiveness. In a specificembodiment, aggressiveness of a tumor is evaluated by determining theextent to which 36P6D5 is expressed in the tumor cells, with higherexpression levels indicating more aggressive tumors. In a closelyrelated embodiment, one can evaluate the integrity of 36P6D5 nucleotideand amino acid sequences in a biological sample in order to identifyperturbations in the structure of these molecules such as insertions,deletions, substitutions and the like, with the presence of one or moreperturbations indicating more aggressive tumors.

The invention additionally provides methods of examining a biologicalsample for evidence of dysregulated cellular growth. In one embodiment,the method comprises comparing the status of 36P6D5 in the biologicalsample to the status of 36P6D5 in a corresponding normal sample, whereinalterations in the status of 36P6D5 in the biological sample areassociated with dysregulated cellular growth. The status of 36P6D5 inthe biological sample can be evaluated by, for example, examining levelsof 36P6D5 mRNA expression or levels of 36P6D5 protein expression. In oneembodiment, an alteration in the status of 36P6D5 is identified by thepresence of 36P6D5 expressing cells in a biological sample from a tissuein which 36P6D5 expressing cells are normally absent.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 36P6D5 mRNA or protein expression in a test cellor tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 36P6D5 mRNA may, for example, beevaluated in tissue samples including but not limited to colon, lung,prostate, pancreas, bladder, breast, ovary, cervix, testis, head andneck, brain, stomach, bone, etc. The presence of significant 36P6D5expression in any of these tissues may be useful to indicate theemergence, presence and/or severity of these cancers or a metastasis ofcancer originating in another tissue, since the corresponding normaltissues do not express 36P6D5 mRNA or express it at lower levels.

Yet another related aspect of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of 36P6D5 mRNA or36P6D5 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 36P6D5 mRNA or 36P6D5 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 36P6D5 mRNA or 36P6D5 proteinexpression in the tumor sample over time provides information on theprogression of the cancer. In a specific embodiment, the progression ofa cancer is evaluated by determining the extent to which 36P6D5expression in the tumor cells alters over time, with higher expressionlevels indicating a progression of the cancer. In a closely relatedembodiment, one can evaluate the integrity 36P6D5 nucleotide and aminoacid sequences in a biological sample in order to identify perturbationsin the structure of these molecules such as insertions, deletions,substitutions and the like, with the presence of one or moreperturbations indicating a progression of the cancer.

The above diagnostic approaches may be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention disclosed herein isdirected to methods for observing a coincidence between the expressionof 36P6D5 gene and 36P6D5 gene products (or perturbations in 36P6D5 geneand 36P6D5 gene products) and a factor that is associated withmalignancy as a means of diagnosing and prognosticating the status of atissue sample. In this context, a wide variety of factors associatedwith malignancy may be utilized such as the expression of genesotherwise associated with malignancy (including PSA, PSCA and PSMexpression) as well as gross cytological observations (see e.g. Bockinget al., 1984, Anal. Quant. Cytol. 6(2):74-88; Eptsein, 1995, Hum.Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51;Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods forobserving a coincidence between the expression of 36P6D5 gene and 36P6D5gene products (or perturbations in 36P6D5 gene and 36P6D5 gene products)and an additional factor that is associated with malignancy are usefulfor example, because the presence of a set or constellation of specificfactors that coincide provides information crucial for diagnosing andprognosticating the status of a tissue sample.

In a typical embodiment, methods for observing a coincidence between theexpression of 36P6D5 gene and 36P6D5 gene products (or perturbations in36P6D5 gene and 36P6D5 gene products) and a factor that is associatedwith malignancy entails detecting the overexpression of 36P6D5 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample, and observing a coincidence of 36P6D5 mRNAor protein and PSA mRNA or protein overexpression. In a specificembodiment, the expression of 36P6D5 and PSA mRNA in prostate tissue isexamined. In a preferred embodiment, the coincidence of 36P6D5 and PSAmRNA overexpression in the sample provides an indication of prostatecancer, prostate cancer susceptibility or the emergence or existence ofa prostate tumor.

Methods for detecting and quantifying the expression of 36P6D5 mRNA orprotein are described herein and use of standard nucleic acid andprotein detection and quantification technologies is well known in theart. Standard methods for the detection and quantification of 36P6D5mRNA include in situ hybridization using labeled 36P6D5 riboprobes,Northern blot and related techniques using 36P6D5 polynucleotide probes,RT-PCR analysis using primers specific for 36P6D5, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment,semi-quantitative RT-PCR may be used to detect and quantify 36P6D5 mRNAexpression as described in the Examples that follow. Any number ofprimers capable of amplifying 36P6D5 may be used for this purpose,including but not limited to the various primer sets specificallydescribed herein. Standard methods for the detection and quantificationof protein may be used for this purpose. In a specific embodiment,polyclonal or monoclonal antibodies specifically reactive with thewild-type 36P6D5 protein may be used in an immunohistochemical assay ofbiopsied tissue.

Identifying Molecules that Interact with 36P6D5

The 36P6D5 protein sequences disclosed herein allow the skilled artisanto identify proteins, small molecules and other agents that interactwith 36P6D5 and pathways activated by 36P6D5 via any one of a variety ofart accepted protocols. For example one can utilize one of the varietyof so-called interaction trap systems (also referred to as the“two-hybrid assay”). In such systems, molecules that interactreconstitute a transcription factor and direct expression of a reportergene, the expression of which is then assayed. Typical systems identifyprotein-protein interactions in vivo through reconstitution of aeukaryotic transcriptional activator and are disclosed for example inU.S. Pat. Nos. 5,955,280, 5,925,523, 5,846,722 and 6,004,746.

Alternatively one can identify molecules that interact with 36P6D5protein sequences by screening peptide libraries. In such methods,peptides that bind to selected receptor molecules such as 36P6D5 areidentified by screening libraries that encode a random or controlledcollection of amino acids. Peptides encoded by the libraries areexpressed as fusion proteins of bacteriophage coat proteins, andbacteriophage particles are then screened against the receptors ofinterest.

Peptides having a wide variety of uses, such as therapeutic ordiagnostic reagents, may thus be identified without any priorinformation on the structure of the expected ligand or receptormolecule. Typical peptide libraries and screening methods that can beused to identify molecules that interact with 36P6D5 protein sequencesare disclosed for example in U.S. Pat. Nos. 5,723,286 and 5,733,731.

Alternatively, cell lines expressing 36P6D5 can be used to identifyprotein-protein interactions mediated by 36P6D5. This possibility can beexamined using immunoprecipitation techniques as shown by others(Hamilton B J, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51).Typically 36P6D5 protein can be immunoprecipitated from 36P6D5expressing prostate cancer cell lines using anti-36P6D5 antibodies.Alternatively, antibodies against His-tag can be used in a cell lineengineered to express 36P6D5 (vectors mentioned above). Theimmunoprecipitated complex can be examined for protein association byprocedures such as western blotting, ³⁵S-methionine labeling ofproteins, protein microsequencing, silver staining and two dimensionalgel electrophoresis.

Small molecules that interact with 36P6D5 can be identified throughrelated embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 36P6D5's ability to mediatephosphorylation and de-phosphorylation, second messenger signaling andtumorigenesis. Typical methods are discussed for example in U.S. Pat.No. 5,928,868 and include methods forming hybrid ligands in which atleast one ligand is a small molecule. In an illustrative embodiment, thehybrid ligand is introduced into cells that in turn contain a first anda second expression vector. Each expression vector includes DNA forexpressing a hybrid protein that encodes a target protein linked to acoding sequence for a transcriptional module. The cells further containsa reporter gene, the expression of which is conditioned on the proximityof the first and second hybrid proteins to each other, an event thatoccurs only if the hybrid ligand binds to target sites on both hybridproteins. Those cells that express the reporter gene are selected andthe unknown small molecule or the unknown hybrid protein is identified.

A typical embodiment of this invention consists of a method of screeningfor a molecule that interacts with a 36P6D5 amino acid sequence shown inFIG. 1 (SEQ ID NO: 2), comprising the steps of contacting a populationof molecules with the 36P6D5 amino acid sequence, allowing thepopulation of molecules and the 36P6D5 amino acid sequence to interactunder conditions that facilitate an interaction, determining thepresence of a molecule that interacts with the 36P6D5 amino acidsequence and then separating molecules that do not interact with the36P6D5 amino acid sequence from molecules that do interact with the36P6D5 amino acid sequence. In a specific embodiment, the method furtherincludes purifying a molecule that interacts with the 36P6D5 amino acidsequence. In a preferred embodiment, the 36P6D5 amino acid sequence iscontacted with a library of peptides.

Using 36P6D5 to Modulate Cellular Microenvironments

A variety of secreted proteins have been described in prostate cancer, anumber of which have been shown to participate in the process of tumorformation and progression (Inoue K Clin Cancer Res. 2000; 6:2104-19, DowJ K, deVere White R W. Urology. 2000; 55:800-6). In the process of tumorprogression, cancer cells to secrete and express molecules that allowthem to grow in both the microenvironment of the local prostate as wellas the bone microenvironment. A unique feature of prostate cancer isthat in the process of tumor progression, cancer cells become metastaticto bone and recent studies suggest that this predilection to metastasizeto the bone is based on the ability of prostate cancer cells to secreteand express molecules that allow them to grow in the bonemicroenvironment (Koeneman K S, Yeung F, Chung L W. Prostate 1999:39:246). In this context, the data presented herein (see e.g. FIGS. 8and 9) suggests that 36P6D5 plays a role (1) in targeting prostate cellsto the bone, (2) allowing the growth of prostate cells in the bonemicroenvironment, (3) inducing the differentiation of prostate tumorcells or bone marrow cells to osteoblasts or (4) supporting theinteraction of bone stroma with prostate cancer cells thereby creating afavorable environment for the growth of cancer cells. Consequently, thismolecule can be used in methods for conditioning media and/or mimickingthe microenvironment in which prostate cancer cells can metastasize.

In order to test the possibility that 36P6D5 interacts with cellsnormally located in the prostate and bone microenvironments, the 36P6D5protein was expressed as a recombinant protein (pTag5 36P6D5). Purifiedrecombinant-36P6D5 was then incubated with a variety of relevant celltypes, including prostate epithelial cells, prostate tumor cell lines,prostate stromal cells, osteosarcoma and bone stromal cells. Binding of36P6D5 to intact cells is detected by FACS analysis and by calorimetricassay. Our studies indicated that, when the recombinant 36P6D5 AP-fusionprotein is incubated in the presence of prostate cancer cells derivedfrom LAPC4 and LAPC9 xenografts, it binds to LAPC9 and LAPC4 xenograftcells. Binding of 36P6D5 to the cell surface was detected usingcalorimetric change in AP substrate (FIG. 8). In contrast, no colorconversion was observed when cells were incubated with the appropriatecontrol. This analysis is valuable as it identifies a cell populationthat binds and may respond to 36P6D5. In addition, the identification ofa target cell population may provide a means of isolating andidentifying 36P6D5 receptors. This information can be used in a varietyof therapeutic application and small molecule design.

Using the data presented herein (see e.g. FIG. 8), one can employmethods to modulate cancer cell phenotypes and dissect the differentstages of metastasis by reproducing the microenvironment of the localprostate in which cancer cells originate as well metastatic cellularmicroenvironment, thereby generating a cancer model in which artisanscan assess novel therapeutic and diagnostic compositions and methods. Anillustrative method consists of modulating the microenvironment of acell by exposing the cell to a 36P6D5 polypeptide so that thepolypeptide binds to the cell, thereby modulating the microenvironmentof the cell. A related method in this context consists of modulating themicroenvironment of a cell by exposing the cell to a molecule thatinteracts with a 36P6D5 polypeptide (such as an anti-36P6D5 antibody)that inhibits or facilitates the binding of the polypeptide to the cell,thereby modulating the microenvironment of the cell. Such methods forgenerating or modulating a specific microenvironmental milieu satisfy aneed in the art to generate a variety diverse microenvironments based onobservations that cancer cells (including prostate cancer cells) aredifferentially regulated depending upon the factors present in the cellmicroenvironment (see e.g. Levesque et al., Endocrinology 139(5):2375-2381 (1998). Consequently the identification 36P6D5 proteins asmolecules which are both secreted by and bind to cells in thismicroenvironment allows the skilled artisan to use this information toinclude and/or manipulate 36P6D in contexts that more faithfullyreproduce and modulate the events occurring in the progression ofcancers.

A number of experiments can designed to investigate how 36P6D5 maycontribute to the growth of prostate cancer cells. In a first typicalset of experiments, prostate cancer epithelial cells are incubated inthe presence or absence of recombinant 36P6D5, and evaluated forproliferation using a well-documented calorimetric assay. In parallel,PC3 cells engineered to stably express 36P6D5 are evaluated for cellgrowth potential. In a second typical set of experiments, taggedprostate cancer cells, such as PC3 cells engineered to express the GreenFluorescence Protein (GFP), are grown in the presence of bone stromalcells. The cells are incubated in the presence or absence of recombinant36P6D5, and evaluated for cell growth by measuring increase in GFP.

Using the disclosure provided herein, it is possible to examine the roleof 36P6D5 in bone metastasis. In order to determine whether 36P6D5induces prostate cells to become osteomimetic, primary prostate cells aswell as cell lines can be grown in the presence or absence ofrecombinant purified 36P6D5. Cells can be then examined for theexpression of early and late markers of bone maturation, includingosteonectin, osteopontin, alkaline phosphatase and osteocalcin. One canalso determine whether 36P6D5 is inducing the expression of growthfactors supportive of prostate cell growth in a traditionally protectivemicroenvironment such as the bone. PCR and ELISA techniques can be usedto investigate the expression and secretion of FGF, HGF and IGF in cellsgrown in the presence or absence of recombinant purified 36P6D5. Similarexperiments can be performed using bone marrow cells to determinewhether 36P6D5 induces the differentiation of chondrocyte progenitors tomature osteocytes. These experiments may be valuable in demonstratingthe role of 36P6D5 in supporting the creation of an environmentfavorable for prostate cancer growth in bone, and identifying targetsfor therapeutic intervention.

Using the disclosure provided herein, it is possible to examine the roleof 36P6D5 in Cell-Cell Interaction. It is possible that 36P6D5 plays arole in recruiting prostate cells to the bone by enhancing prostate cellinteraction with stromal cells or osteocytes. GFP expressing prostatecancer cells can be grown in the presence or absence of recombinant36P6D5 protein. GPF cells can be incubated with control or 36P6D5treated stromal cells and osteocytes for various amounts of time.Non-adherent cells can be removed and adhesion to stroma and osteocytescan be evaluated by measuring the amount of GFP in the culture. Thisdata will be critical in considering inhibitors of factors whichmodulate the microenvironment of the local prostate in which cancercells originate and grow as the colonization of metastatic sites bycancer cells.

Therapeutic Methods and Compositions

The identification of 36P6D5 as a gene that is highly expressed incancers of the prostate (and possibly other cancers), opens a number oftherapeutic approaches to the treatment of such cancers. As discussedabove, it is possible that 36P6D5 is secreted from cancer cells and inthis way modulates proliferation signals. Its potential role as atranscription factor and its high expression in prostate cancer makes ita potential target for small molecule-mediated therapy.

Accordingly, therapeutic approaches aimed at inhibiting the activity ofthe 36P6D5 protein are expected to be useful for patients suffering fromprostate cancer and other cancers expressing 36P6D5. These therapeuticapproaches aimed at inhibiting the activity of the 36P6D5 proteingenerally fall into two classes. One class comprises various methods forinhibiting the binding or association of the 36P6D5 protein with itsbinding partner or with other proteins. Another class comprises avariety of methods for inhibiting the transcription of the 36P6D5 geneor translation of 36P6D5 mRNA.

36P6D5 as a Target for Antibody-Based Therapy

The structural features of 36P6D5 indicate that this molecule is anattractive target for antibody-based therapeutic strategies. A number oftypical antibody strategies are known in the art for targeting bothextracellular and intracellular molecules (see e.g. complement and ADCCmediated killing as well as the use of intrabodies discussed below).Because 36P6D5 is expressed by cancer cells of various lineages and notby corresponding normal cells, systemic administration of36P6D5-immunoreactive compositions would be expected to exhibitexcellent sensitivity without toxic, non-specific and/or non-targeteffects caused by binding of the immunotherapeutic molecule tonon-target organs and tissues. Antibodies specifically reactive withdomains of 36P6D5 can be useful to treat 36P6D5-expressing cancerssystemically, either as conjugates with a toxin or therapeutic agent, oras naked antibodies capable of inhibiting cell proliferation orfunction.

36P6D5 antibodies can be introduced into a patient such that theantibody binds to 36P6D5 and modulates or perturbs a function such as aninteraction with a binding partner and consequently mediates the growthinhibition and/or destruction of the cells and the tumor and/or inhibitsthe growth of the cells or the tumor. Mechanisms by which suchantibodies exert a therapeutic effect may include complement-mediatedcytolysis, antibody-dependent cellular cytotoxicity, modulating thephysiological function of 36P6D5, inhibiting ligand binding or signaltransduction pathways, modulating tumor cell differentiation, alteringtumor angiogenesis factor profiles, and/or by inducing apoptosis. 36P6D5antibodies can be conjugated to toxic or therapeutic agents and used todeliver the toxic or therapeutic agent directly to 36P6D5-bearing tumorcells. Examples of toxic agents include, but are not limited to,calchemicin, maytansinoids, radioisotopes such as ¹³¹I, ytrium, andbismuth.

Cancer immunotherapy using anti-36P6D5 antibodies may follow theteachings generated from various approaches that have been successfullyemployed in the treatment of other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol.18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186;Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk etal., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al.,1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhonget al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20antibodies (e.g., Rituxan™, IDEC Pharmaceuticals Corp.), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Fortreatment of prostate cancer, for example, 36P6D5 antibodies can beadministered in conjunction with radiation, chemotherapy or hormoneablation.

Although 36P6D5 antibody therapy may be useful for all stages of cancer,antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionmay be indicated for patients who have received previously one or morechemotherapy, while combining the antibody therapy of the invention witha chemotherapeutic or radiation regimen may be preferred for patientswho have not received chemotherapeutic treatment. Additionally, antibodytherapy may enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

It may be desirable for some cancer patients to be evaluated for thepresence and level of 36P6D5 expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative 36P6D5imaging, or other techniques capable of reliably indicating the presenceand degree of 36P6D5 expression. Immunohistochemical analysis of tumorbiopsies or surgical specimens may be preferred for this purpose.Methods for immunohistochemical analysis of tumor tissues are well knownin the art.

Anti-36P6D5 monoclonal antibodies useful in treating prostate and othercancers include those that are capable of initiating a potent immuneresponse against the tumor and those that are capable of directcytotoxicity. In this regard, anti-36P6D5 monoclonal antibodies (mAbs)may elicit tumor cell lysis by either complement-mediated orantibody-dependent cell cytotoxicity (ADCC) mechanisms, both of whichrequire an intact Fc portion of the immunoglobulin molecule forinteraction with effector cell Fc receptor sites or complement proteins.In addition, anti-36P6D5 mAbs that exert a direct biological effect ontumor growth are useful in the practice of the invention. Potentialmechanisms by which such directly cytotoxic mAbs may act includeinhibition of cell growth, modulation of cellular differentiation,modulation of tumor angiogenesis factor profiles, and the induction ofapoptosis. The mechanism by which a particular anti-36P6D5 mAb exerts ananti-tumor effect may be evaluated using any number of in vitro assaysdesigned to determine ADCC, ADMMC, complement-mediated cell lysis, andso forth, as is generally known in the art.

The use of murine or other non-human monoclonal antibodies, orhuman/mouse chimeric mAbs may induce moderate to strong immune responsesin some patients. In some cases, this will result in clearance of theantibody from circulation and reduced efficacy. In the most severecases, such an immune response may lead to the extensive formation ofimmune complexes which, potentially, can cause renal failure.Accordingly, preferred monoclonal antibodies used in the practice of thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 36P6D5antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-36P6D5 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails may have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination mayexhibit synergistic therapeutic effects. In addition, the administrationof anti-36P6D5 mAbs may be combined with other therapeutic agents,including but not limited to various chemotherapeutic agents,androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF). Theanti-36P6D5 mAbs may be administered in their “naked” or unconjugatedform, or may have therapeutic agents conjugated to them.

The anti-36P6D5 antibody formulations may be administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumor, intradermal,and the like. Treatment will generally involve the repeatedadministration of the anti-36P6D5 antibody preparation via an acceptableroute of administration such as intravenous injection (IV), typically ata dose in the range of about 0.1 to about 10 mg/kg body weight. Doses inthe range of 10-500 mg mAb per week may be effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti-36P6D5 mAb preparation may represent an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose may beadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. However, as one of skill in the art willunderstand, various factors will influence the ideal dose regimen in aparticular case. Such factors may include, for example, the bindingaffinity and half life of the Ab or mAbs used, the degree of 36P6D5expression in the patient, the extent of circulating shed 36P6D5antigen, the desired steady-state antibody concentration level,frequency of treatment, and the influence of chemotherapeutic agentsused in combination with the treatment method of the invention.

Optimally, patients should be evaluated for the level of circulatingshed 36P6D5 antigen in serum in order to assist in the determination ofthe most effective dosing regimen and related factors. Such evaluationsmay also be used for monitoring purposes throughout therapy, and may beuseful to gauge therapeutic success in combination with evaluating otherparameters (such as serum PSA levels in prostate cancer therapy).

Inhibition of 36P6D5 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 36P6D5 to its binding partner or ligand, or itsassociation with other protein(s) as well as methods for inhibiting36P6D5 function.

Inhibition of 36P6D5 with Intracellular Antibodies

In one approach, recombinant vectors encoding single chain antibodiesthat specifically bind to 36P6D5 may be introduced into 36P6D5expressing cells via gene transfer technologies, wherein the encodedsingle chain anti-36P6D5 antibody is expressed intracellularly, binds to36P6D5 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, may bespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatmentwill be focused. This technology has been successfully applied in theart (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).Intrabodies have been shown to virtually eliminate the expression ofotherwise abundant cell surface receptors. See, for example, Richardsonet al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.1: 332-337.

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies may beexpressed as a single chain variable region fragment joined to the lightchain constant region. Well known intracellular trafficking signals maybe engineered into recombinant polynucleotide vectors encoding suchsingle chain antibodies in order to precisely target the expressedintrabody to the desired intracellular compartment. For example,intrabodies targeted to the endoplasmic reticulum (ER) may be engineeredto incorporate a leader peptide and, optionally, a C-terminal ERretention signal, such as the KDEL amino acid motif Intrabodies intendedto exert activity in the nucleus may be engineered to include a nuclearlocalization signal. Lipid moieties may be joined to intrabodies inorder to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies may also be targeted to exert function in thecytosol. For example, cytosolic intrabodies may be used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

In one embodiment, intrabodies may be used to capture 36P6D5 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals may be engineered into such 36P6D5 intrabodies inorder to achieve the desired targeting. Such 36P6D5 intrabodies may bedesigned to bind specifically to a particular 36P6D5 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to the 36P6D5protein may be used to prevent 36P6D5 from gang access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 36P6D5 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular tumor cells, the transcription of the intrabody may be placedunder the regulatory control of an appropriate tumor-specific promoterand/or enhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer may beutilized (See, for example, U.S. Pat. No. 5,919,652).

Inhibition of 36P6D5 with Recombinant Proteins

In another approach, recombinant molecules that are capable of bindingto 36P6D5 thereby preventing 36P6D5 from accessing/binding to itsbinding partner(s) or associating with other protein(s) are used toinhibit 36P6D5 function. Such recombinant molecules may, for example,contain the reactive part(s) of a 36P6D5 specific antibody molecule. Ina particular embodiment, the 36P6D5 binding domain of a 36P6D5 bindingpartner may be engineered into a dimeric fusion protein comprising two36P6D5 ligand binding domains linked to the Fc portion of a human IgG,such as human IgG1. Such IgG portion may contain, for example, theC_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1domain. Such dimeric fusion proteins may be administered in soluble formto patients suffering from a cancer associated with the expression of36P6D5, including but not limited to prostate, bladder, ovarian, breast,pancreas, colon and lung cancers, where the dimeric fusion proteinspecifically binds to 36P6D5 thereby blocking 36P6D5 interaction with abinding partner. Such dimeric fusion proteins may be further combinedinto multimeric proteins using known antibody linking technologies.

Inhibition of 36P6D5 Transcription or Translation

Within another class of therapeutic approaches, the invention providesvarious methods and compositions for inhibiting the transcription of the36P6D5 gene. Similarly, the invention also provides methods andcompositions for inhibiting the translation of 36P6D5 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 36P6D5gene comprises contacting the 36P6D5 gene with a 36P6D5 antisensepolynucleotide. In another approach, a method of inhibiting 36P6D5 mRNAtranslation comprises contacting the 36P6D5 mRNA with an antisensepolynucleotide. In another approach, a 36P6D5 specific ribozyme may beused to cleave the 36P6D5 message, thereby inhibiting translation. Suchantisense and ribozyme based methods may also be directed to theregulatory regions of the 36P6D5 gene, such as the 36P6D5 start site,promoter and/or enhancer elements. Similarly, proteins capable ofinhibiting a 36P6D5 gene transcription factor may be used to inhibit36P6D5 mRNA transcription. The various polynucleotides and compositionsuseful in the aforementioned methods have been described above. Inanother approach, one can inhibit the translation of the 36P6D6 geneusing morpholino antisense technology. The use of antisense and ribozymemolecules to inhibit transcription and translation is well known in theart.

Other factors that inhibit the transcription of 36P6D5 throughinterfering with 36P6D5 transcriptional activation may also be usefulfor the treatment of cancers expressing 36P6D5. Similarly, factors thatare capable of interfering with 36P6D5 processing may be useful for thetreatment of cancers expressing 36P6D5. Cancer treatment methodsutilizing such factors are also within the scope of the invention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies may be used for deliveringtherapeutic polynucleotide molecules to tumor cells synthesizing 36P6D5(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 36P6D5 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 36P6D5 antisensepolynucleotides, ribozymes, factors capable of interfering with 36P6D5transcription, and so forth, may be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches may be combined with any one of a widevariety of chemotherapy or radiation therapy regimens. These therapeuticapproaches may also enable the use of reduced dosages of chemotherapyand/or less frequent administration, particularly in patients that donot tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, may beevaluated using various in vitro and in vivo assay systems. In vitroassays for evaluating therapeutic potential include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 36P6D5 to a bindingpartner, etc.

In vivo, the effect of a 36P6D5 therapeutic composition may be evaluatedin a suitable animal model. For example, xenogenic prostate cancermodels wherein human prostate cancer explants or passaged xenografttissues are introduced into immune compromised animals, such as nude orSCID mice, are appropriate in relation to prostate cancer and have beendescribed (Klein et al., 1997, Nature Medicine 3:402-408). For example,PCT Patent Application WO98/16628, Sawyers et al., published Apr. 23,1998, describes various xenograft models of human prostate cancercapable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy may be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like. See, also, the Examples below.

In vivo assays that qualify the promotion of apoptosis may also beuseful in evaluating potential therapeutic compositions. In oneembodiment, xenografts from bearing mice treated with the therapeuticcomposition may be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isnon-reactive with the patient's immune system. Examples include, but arenot limited to, any of a number of standard pharmaceutical carriers suchas sterile phosphate buffered saline solutions, bacteriostatic water,and the like (see, generally, Remington's Pharmaceutical Sciences16^(th) Ed., A. Osal., Ed., 1980).

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations may be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancerand will generally depend on a number of other factors appreciated inthe art.

Cancer Vaccines

As noted above, the expression profile of 36P6D5 shows that it is highlyexpressed in advanced and metastasized prostate cancer. This expressionpattern is reminiscent of the Cancer-Testis (CT) antigens or MAGEs,which are testis-specific genes that are up-regulated in melanomas andother cancers (Van den Eynde and Boon, Int J Clin Lab Res. 27:81-86,1997). Due to their tissue-specific expression and high expressionlevels in cancer, the MAGEs are currently being investigated as targetsfor cancer vaccines (Durrant, Anticancer Drugs 8:727-733, 1997; Reynoldset al., Int J Cancer 72:972-976, 1997).

The invention further provides cancer vaccines comprising a 36P6D5protein or fragment thereof, as well as DNA based vaccines. In view ofthe expression of 36P6D5 cancer vaccines are expected to be effective atspecifically preventing and/or treating 36P6D5 expressing cancerswithout creating non-specific effects on non-target tissues. The use ofa tumor antigen in a vaccine for generating humoral and cell-mediatedimmunity for use in anti-cancer therapy is well known in the art and hasbeen employed in prostate cancer using human PSMA and rodent PAPimmunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al.,1997, J. Immunol. 159:3113-3117). Such methods can be readily practicedby employing a 36P6D5 protein, or fragment thereof, or a 36P6D5-encodingnucleic acid molecule and recombinant vectors capable of expressing andappropriately presenting the 36P6D5 immunogen. An illustrative exampleof a typical technique consists of a method of generating an immuneresponse (e.g. a humoral response) in a mammal comprising the stepsexposing the mammal's immune system to an immunoreactive epitope (e.g.an epitope of the 36P6D5 protein shown in SEQ ID NO: 2) so that themammal generates an immune response that is specific for that epitope isgenerated (e.g. antibodies that specifically recognize that epitope).

For example, viral gene delivery systems may be used to deliver a36P6D5-encoding nucleic acid molecule. Various viral gene deliverysystems that can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8:658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a 36P6D5 protein or fragment thereof introduced into thepatient (e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human 36P6D5 cDNA may be employed. Inanother embodiment, 36P6D5 nucleic acid molecules encoding specificcytotoxic T lymphocyte (CTL) epitopes may be employed. CTL epitopes canbe determined using specific algorithms (e.g., Epimer, Brown University)to identify peptides within a 36P6D5 protein that are capable ofoptimally binding to specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present 36P6D5 antigen to a patient'simmune system. Dendritic cells express MHC class I and II, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al.,1996, Prostate 29:371-380). Dendritic cells can be used to present36P6D5 peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed with36P6D5 peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete 36P6D5 protein.Yet another embodiment involves engineering the overexpression of the36P6D5 gene in dendritic cells using various implementing vectors knownin the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther.4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770),lentivirus, adeno-associated virus, DNA transfection (Ribas et al.,1997, Cancer Res. 57:2865-2869), and tumor-derived RNA transfection(Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells expressing36P6D5 may also be engineered to express immune modulators, such asGM-CSF, and used as immunizing agents.

Anti-idiotypic anti-36P6D5 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 36P6D5 protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-36P6D5 antibodies that mimic an epitope ona 36P6D5 protein (see, for example, Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al.,1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypicantibody can be used in cancer vaccine strategies.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing 36P6D5. Constructs comprising DNA encoding a36P6D5 protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded 36P6D5 protein/immunogen. Expression of the 36P6D5 proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against bone, colon, pancreatic prostate,kidney, bladder and ovarian cancers. Various prophylactic andtherapeutic genetic immunization techniques known in the art may be used(for review, see information and references published at Internetaddress www.genweb.com).

Diagnostic Compositions and Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a 36P6D5 proteinor a 36P6D5 gene or message, respectively. Where the kit utilizesnucleic acid hybridization to detect the target nucleic acid, the kitmay also have containers containing nucleotide(s) for amplification ofthe target nucleic acid sequence and/or a container comprising areporter-means, such as a biotin-binding protein, such as avidin orstreptavidin, bound to a reporter molecule, such as an enzymatic,florescent, or radioisotope label.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

Accordingly, the invention also provides diagnostic compositionscomprising 36P6D5-related molecules. Such molecules include the various36P6D5 polynucleotides, primers, probes, proteins, fragments, antibodiesdescribed herein. The molecules included in the diagnostic compositionmay optionally be labeled with a detectable marker. 36P6D5 diagnosticcompositions may further comprise appropriate buffers, diluents, andother ingredients as desired.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples which follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the 36P6D5 Gene

Materials and Methods

LAPC Xenografts:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402-408).Androgen dependent LAPC-4 xenografts (LAPC-4 AD) were grownsubcutaneously in male SCID mice and were passaged as small tissuechunks in recipient males. LAPC-4 AD xenografts were grown intratibiallyas follows. LAPC-4 AD xenograft tumor tissue grown subcutaneously wasminced into 1-2 mm³ sections while the tissue was bathed in 1× Iscovesmedium, minced tissue was then centrifuged at 1.3K rpm for 4 minutes,the supernatant was resuspended in 10 ml ice cold 1× Iscoves medium andcentrifuged at 1.3K rpm for 4 minutes. The pellet was then resuspendedin 1× Iscoves with 1% pronase E and incubated for 20 minutes at roomtemperature with mild rocking agitation followed by incubation on icefor 2-4 minutes. Filtrate was centrifuged at 1.3K rmp for 4 minutes, andthe pronase was removed from the aspirated pellet by resuspending in 10ml Iscoves and re-centrifuging. Clumps of cells were then plated inPrEGM medium and grown overnight. The cells were then harvested,filtered, washed 2×RPMI, and counted. Approximately 50,000 cells weremixed with and equal volume of ice-cold Matrigel on ice, and surgicallyinjected into the proximal tibial metaphyses of SCID mice via a 27 gaugeneedle. After 10-12 weeks, LAPC-4 tumors growing in bone marrow wererecovered.

Cell Lines:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): (SEQ ID NO: 3) 5′TTTGATCAAGCTT₃₀3′Adaptor 1: (SEQ ID NO: 4) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′(SEQ ID NO: 5) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 6)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 7)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 8) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO: 9) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nestedprimer (NP)2: (SEQ ID NO: 10) 5′AGCGTGGTCGCGGCCGAGGA3′Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes which may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from LAPC-4 AD xenografts growingin two different environments, namely the subcutaneous (“LAPC-4 AD SQ”)and intratibial (“LAPC-4 AD IT”) growth environments, wherein the LAPC-4AD IT xenograft was used as the source of the “tester” cDNA, while theLAPC-4 AD SQ xenograft was used as the source of the “driver” cDNA.

Double stranded cDNAs corresponding to tester and driver cDNAs weresynthesized from 2 μg of poly(A)⁺ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hrs. at 37° C.Digested cDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining Dpn II digested cDNA from thehuman cell lines HeLa, 293, A431, Colo205, and mouse liver. Tester cDNAwas generated by diluting 1 μl of Dpn II digested cDNA from the relevantxenograft source (see above) (400 ng) in 5 μl of water. The diluted cDNA(2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10μM), in separate ligation reactions, in a total volume of 10 μl at 16°C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation wasterminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM) 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 μl of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12-18priming using the Gibco-BRL Superscript Preamplification system. Themanufacturers protocol was used and included an incubation for 50 min at42° C. with reverse transcriptase followed by RNAse H treatment at 37°C. for 20 min. After completing the reaction, the volume was increasedto 200 μl with water prior to normalization. First strand cDNAs from 16different normal human tissues were obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:11) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 12) to amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction was removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: initial denaturation was at 94° C. for 15 sec, followed by a18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 bp β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalβ-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization were required to achieve equal band intensitiesin all tissues after 22 cycles of PCR.

To determine expression levels of the 36P6D5 gene, 5 μl of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs:

36P6D5.1 GCATTCTTGGCATCGTTATTCAG (SEQ ID NO: 13) 36P6D5.2TAACTGGGAATGTGACAGCAACAC (SEQ ID NO: 14)

Semi quantitative expression analysis was achieved by comparing the PCRproducts at cycle numbers that give light band intensities.

Results:

The SSH experiment described in the Materials and Methods, supra, led tothe isolation of numerous candidate gene fragment clones (SSH clones).All candidate clones were sequenced and subjected to homology analysisagainst all sequences in the major public gene and EST databases inorder to provide information on the identity of the corresponding geneand to help guide the decision to analyze a particular gene fordifferential expression. In general, gene fragments which had nohomology to any known sequence in any of the searched databases, andthus considered to represent novel genes, as well as gene fragmentsshowing homology to previously sequenced expressed sequence tags ESTs),were subjected to differential expression analysis by RT-PCR and/orNorthern analysis.

One of the SHH clones comprising about 423 bp, showed no homology to anyknown gene, and was designated 36P6D5. Initial expression analysis of36P6D5 by RT-PCR showed highest expression in LAPC-4 AD (IT) and normalprostate compared to the other samples. This clone, therefore, wasutilized for obtaining a full length cDNA encoding 36P6D5 as describedin Example 2, below.

Example 2 Isolation of Full Length cDNA Encoding the 36P6D5 Gene

The isolated 36P6D5 gene fragment of 423 bp was used as a probe toidentify the full length cDNA for 36P6D5 in a human prostate cDNAlibrary. This resulted in the isolation of a 931 bp cDNA, clone36P6D5-GTC4, which encodes a 235 amino acid ORF with significanthomology to two previously reported sequences, the 2-19 proteinprecursor (Genbank P98173) and a gene isolated from human osteoblaststermed GS3786 (Q92520).

The nucleotide and deduced amino acid sequences of the clone 36P6D5-GTC4cDNA are shown in FIG. 1. The encoded amino acid sequence exhibits anN-terminal signal sequence, which predicts the protein to be secreted(using the PSORT program). amino Acid sequence alignments of the 36P6D5protein with 2-19 protein precursor and osteoblast protein GS3786 areshown in FIG. 2.

Example 3 Northern Blot Analysis of 36P6D5 Gene Expression

36P6D5 mRNA expression in normal human tissues was first analyzed byNorthern blotting two multiple tissue blots obtained from Clontech (PaloAlto, Calif.), comprising a total of 16 different normal human tissues,using labeled 36P6D5 cDNA as a probe. RNA samples were quantitativelynormalized with a β-actin probe. The results are shown in FIG. 3 andindicate that, within the 16 tissues tested, the 36P6D5 gene ispredominantly expressed in pancreas, with very low level expression alsodetected in prostate and small intestine.

In addition, in order to analyze 36P6D5 expression in human cancertissues and cell lines, RNAs derived from LAPC-4 human prostate cancerxenografts and a panel of non-prostate cancer cell lines were analyzedby Northern blot using the 36P6D5 cDNA as probe. All RNA samples werequantitatively normalized by ethidium bromide staining and subsequentanalysis with a labeled β-actin probe. The results of this analysis arepresented in FIG. 4, and show 36P6D5 expression in LAPC-4 prostatecancer xenografts growing subcutaneously and intratibially, in all casesat higher levels relative to normal prostate. Additionally, significantexpression was detected in several non-prostate cancer cell linesincluding pancreatic (Capan-1), colon (CaCo-2, Colo-205), breast(CAMA-1, DU4475), and ovarian (SW626, CAOV-3, OV1063) cancer cells, alsoat high levels in some cases. In particular, the highest level ofexpression was detected in the breast cancer cell line DU4475.

Example 4 Production and Purification of Recombinant 36P6D5

To express recombinant 36P6D5 for use in a number of contexts such asanalyzing the subcellular localization of 36P6D5 protein, a partial orthe full length cDNA can cloned into any one of a variety of expressionvectors such as those that provide a 6H is tag at the carboxyl-terminus(e.g. pcDNA 3.1 myc-his, InVitrogen).

In a typical embodiment, in order to drive high level expression of36P6D5 protein, the 36P6D5 cDNA encoding amino acids 30-235 (minusN-terminal signal sequence) was cloned into the pAPTag5 mammaliansecretion vector (GenHunter) with and without fusion to the providedalkaline phosphatase (AP) cDNA sequence. This vector provides aC-terminal 6×His and MYC tag for purification and detection and anN-terminal Ig leader sequence to drive secretion. 293T cells stablyexpressing either pAPTag5-36P6D5 or pTag5-36P6D5 (not fused to AP) serveas a source of recombinant protein for purification as visualized by ananti-His Western blot of conditioned media from these cell lines (FIG.5). The HIS-tagged 36P6D5 proteins present in the conditioned media arepurified using the following method. Conditioned media is concentrated5-10 fold and simultaneously buffer exchanged into a phosphate buffer(pH 8.0) containing 500 mM, NaCl and 20 mM imidazole (buffer A) using anamicon ultrafiltration unit with a 10 kd MW cutoff membrane. The prep isbatch bound to 0.1 to 0.5 ml of nickel metal affinity resin (Ni-NTA,Qiagen) and washed extensively with buffer A. The HIS-taggedSGP-28/CRISP-3 protein is then eluted with a 0 to 400 mM gradient ofimidazole in phosphate buffer (pH 6.3) containing 300 mM NaCl and thendialyzed extensively against PBS. The purified protein may then be usedfor growth assays, ligand binding studies, or as immunogen forgenerating antibody reagents.

Additional embodiments of typical constructs are provided below.

pcDNA3.1/MycHis Construct

To express 36P6D5 in mammalian cells, the 705 bp (235 amino acid) 36P6D5ORF was cloned into pcDNA3.1/MycHis_Version A (Invitrogen, Carlsbad,Calif.). Protein expression is driven from the cytomegalovirus (CMV)promoter. The recombinant protein has the myc and six histidines fusedto the C-terminus. The pcDNA3.1/MycHis vector also contains the bovinegrowth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells expressing the protein and the ampicillinresistance gene and ColE1 origin permits selection and maintenance ofthe plasmid in E. coli.

pAPtag

The 36P6D5 protein without the signal sequence (amino acids 30 to 235)was cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). Thisconstruct generates an alkaline phosphatase fusion at the C-terminus ofthe 36P6D5 protein while fusing the IgGK signal sequence to N-terminus.The resulting recombinant 36P6D5 protein is optimized for secretion intothe media of transfected mammalian cells and can be used to identifyproteins such as ligands or receptors that interact with the 36P6D5protein. Protein expression is driven from the CMV promoter and therecombinant protein also contains myc and six histidines fused to theC-terminus of alkaline phosphatase. The Zeosin resistance gene allowsfor selection of mammalian cells expressing the protein and theampicillin resistance gene permits selection of the plasmid in E. coli.

ptag5

The 36P6D5 protein without the signal sequence (amino acids 30 to 235)was also cloned into pTag-5. This vector is similar to pAPTag butwithout the alkaline phosphatase fusion.

pSRa Constructs

To generate mammalian cell lines expressing 36P6D5 constitutively, the705 bp (235 amino acid) ORF was cloned into pSRa constructs. Amphotropicand ecotropic retroviruses are generated by transfection of pSRaconstructs into the 293T-10A1 packaging line or co-transfection of pSRaand a helper plasmid (φ□) in 293 cells, respectively. The retrovirus canbe used to infect a variety of mammalian cell lines, resulting in theintegration of the cloned gene, 36P6D5, into the host cell-lines.Protein expression is driven from a long terminal repeat (LTR). TheNeomycin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli. Anadditional pSRa construct was made that fused the FLAG tag to theC-terminus to allow detection using anti-FLAG antibodies. The FLAGnucleotide sequence was added to cloning primer at the 3′ end of theORF.

Additional pSRa constructs can be made to produce both N-terminal andC-terminal GFP and myc/6 HIS fusion proteins of the full-length 36P6D5protein.

Example 5 Production of Recombinant 36P6D5 in a Baculovirus System

To generate recombinant 36P6D5 protein in a baculovirus expressionsystem, 36P6D5 cDNA is cloned into the baculovirus transfer vectorpBlueBac 4.5 (Invitrogen) which provides a His-tag at the N-terminus.Specifically, pBlueBac-36P6D5 is co-transfected with helper plasmidpBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cellsto generate recombinant baculovirus (see Invitrogen instruction manualfor details). Baculovirus is then collected from cell supernatant andpurified by plaque assay.

Recombinant 36P6D5 protein is then generated by infection of HighFiveinsect cells (InVitrogen) with the purified baculovirus. Recombinant36P6D5 protein may be detected using 36P6D5-specific antibody. 36P6D5protein may be purified and used in various cell based assays or asimmunogen to generate polyclonal and monoclonal antibodies specific for36P6D5.

Example 6 Generation of 36P6D5 Polyclonal Antibodies

To generate polyclonal sera to 36P6D5 a peptide was synthesizedcorresponding to amino acids 163-177 (MVTYDDGSTRLNNDA) (SEQ ID NO: 12)of the SGP-28/CRISP-3 protein sequence was coupled to Keyhole limpethemacyanin (KLH) and was used to immunize a rabbit as follows. Therabbit was initially immunized with 200 ug of peptide-KLH mixed incomplete Freund's adjuvant. The rabbit was then injected every two weekswith 200 ug of peptide-KLH in incomplete Freund's adjuvant. Bleeds weretaken approximately 7-10 days following each immunization. ELISA andWestern blotting analyses were used to determine specificity and titerof the rabbit serum to the immunizing peptide and 36P6D5 proteinrespectively. Affinity purified 36P6D5 polyclonal antibodies wereprepared by passage of crude serum from immunized rabbit over anaffinity matrix comprised of 36P6D5 peptide (MVTYDDGSTRLNNDA) (aminoacids 163-177 of SEQ ID NO: 2) covalently coupled to Affigel 15(BioRad). After extensive washing of the matrix with PBS, antibodiesspecific to 36P6D5 peptide were eluted with low pH glycine buffer (0.1M,pH 2.5), immediately neutralized, and extensively dialyzed against PBS.

To test the rabbit polyclonal antibody for reactivity with 36P6D5protein, Western blot analysis was carried out against conditioned mediaof 293T cells transfected with the pAPTag5-36P6D5 or PTag5-36P6D5expression vectors. Affinity purified rabbit anti-36P6D5 pAb (1 μg/ml)recognizes both forms of recombinant 36P6D5 protein secreted from 293Tcells (FIG. 5).

Example 7 Generation of 36P6D5 Monoclonal Antibodies

In order to generate 36P6D5 monoclonal antibodies, purified293T-expressed HIS-tagged 36P6D5 protein is used to immunize Balb/Cmice. Balb C mice are initially immunized intraperitoneally with 50 μgof 36P6D5 protein mixed in complete Freund's adjuvant. Mice aresubsequently immunized every 2 weeks with 50 μg of 36P6D5 protein mixedin Freund's incomplete adjuvant for a total of 3 immunizations.Reactivity of serum from immunized mice to full length 36P6D5 protein ismonitored by ELISA using the immunogen and by Western blot usingconditioned media from cells expressing 36P6D5 protein. Mice showing thestrongest reactivity are rested for 3 weeks and given a final injectionof fusion protein in PBS and then sacrificed 4 days later. The spleensof the sacrificed mice are then harvested and fused to SPO/2 myelomacells or other suitable myeloma fusion partner using standard procedures(Harlow and Lane, 1988). Supernatants from growth wells following HATselection are screened by ELISA and Western blot to identify 36P6D5specific antibody producing clones.

The binding affinity of a 36P6D5 monoclonal antibody may be determinedusing standard technology. Affinity measurements quantify the strengthof antibody to epitope binding and may be used to help define which36P6D5 monoclonal antibodies are preferred for diagnostic or therapeuticuse. The BIAcore system (Uppsala, Sweden) is a preferred method fordetermining binding affinity. The BIAcore system uses surface plasmonresonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton andMyszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecularinteractions in real time. BIAcore analysis conveniently generatesassociation rate constants, dissociation rate constants, equilibriumdissociation constants, and affinity constants.

In a specific illustration of a typical method for generating 36P6D5monoclonal antibodies, purified 293T-expressed HIS-tagged 36P6D5 proteinwas used to immunize 3 female Balb C mice. Balb C mice were initiallyimmunized intraperitoneally (IP) with 50 μg of 36P6D5 protein mixed inRibi adjuvant and boosted 2 additional times IP in 2 week intervals with25 ug of 36P6D5 protein in Ribi adjuvant. Following the third boost,mice were subsequently immunized with 25 ug of protein IP in PBS. ELISAanalysis of test bleeds following the fourth immunization indicated atiter of at least 2×10⁶ for each of the immunized mice toward theimmunogen. The serum from the immunized mice specifically recognizeendogenous 36P6D5 protein in cell lysates and conditioned media from avariety of cancer cell lines including cell lines derived from prostate(LAPC4 xenograft), colon (Colo 205, CaCo-2), breast (DU4475), andpancreatic (Capan-1) cancers (FIG. 9). In addition, purified polyclonalantibodies from the sera were used to develop a capture ELISA thatspecifically detects 36P6D5 protein in the supernatants of PC-3-36P6D5and DU4475 cell lines and in clinical serum samples (Table 2). Forgeneration of anti-36P6D5 hybridomas, mice are given a final boost of 25ug of 36P6D5 IP, sacrificed 3 days later and harvested spleen cells arefused to myeloma partners using standard procedures (Harlow and Lane,1988). Supernatants from fused hybridoma growth wells are screened byELISA and Western blot to identify 36P6D5 specific antibody producingclones.

Using these antibodies, 36P6D5 protein was detected in several cancercell lines including those derived from colon, pancreas, and breast, andin prostate cancer xenografts (FIG. 9). In addition, 36P6D5 protein wasdetected in conditioned medium of cells that express the proteinendogenously, demonstrating that it is a secreted protein and that itmay serve as a diagnostic serum marker. Indeed, using a sensitivecapture ELISA (FIG. 10), 36P6D5 protein was detected in 7/10 coloncancer samples and 6/10 pancreatic cancer samples, but only 1/6 normalmale samples (Table 2). These results provide evidence that 36P6D5 mayserve as a diagnostic and possibly a therapeutic target for colon andpancreatic cancer as well as other cancers including those derived fromprostate and breast tissues.

Example 8 Identification of Potential Signal Transduction Pathways

To determine whether 36P6D5 directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressing36P6D5. These transcriptional reporters contain consensus binding sitesfor known transcription factors which lie downstream of wellcharacterized signal transduction pathways. The reporters and examplesof there associated transcription factors, signal transduction pathways,and activation stimuli are listed below.

-   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress-   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation-   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress-   4. ARE-luc, androgen receptor; steroids/MAPK;    growth/differentiation/apoptosis-   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis-   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

36P6D5-mediated effects may be assayed in cells showing mRNA expression,such as the 36P6D5-expressing cancer cell lines shown in FIG. 4.Luciferase reporter plasmids may be introduced by lipid mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellsextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Example 9 In Vitro Assays of 36P6D5 Function

The expression profile of 36P6D5 in cancer suggests a functional role intumor initiation, progression and/or maintenance. 36P6D5 may function asa secreted factor that stimulates the proliferation of prostate cancercells in bone. 36P6D5 function can be assessed in mammalian cells usingin vitro approaches. For mammalian expression, 36P6D5 can be cloned intoa number of appropriate vectors, including pcDNA 3.1 myc-His-tag and theretroviral vector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Usingsuch expression vectors, 36P6D5 can be expressed in several cancer celllines, including for example PC-3, NIH 3T3, LNCaP and 293T. Expressionof 36P6D5 can be monitored using anti-36P6D5 antibodies.

Mammalian cell lines expressing 36P6D5 can be tested in several in vitroand in vivo assays, including cell proliferation in tissue culture,activation of apoptotic signals, primary and metastatic tumor formationin SCID mice, and in vitro invasion using a membrane invasion culturesystem (MICS) (Welch et al., Int. J. Cancer 43: 449-457). 36P6D5 cellphenotype is compared to the phenotype of cells that lack expression of36P6D5.

Cell lines expressing 36P6D5 can also be assayed for alteration ofinvasive and migratory properties by measuring passage of cells througha matrigel coated porous membrane chamber (Becton Dickinson). Passage ofcells through the membrane to the opposite side is monitored using afluorescent assay (Becton Dickinson Technical Bulletin #428) usingcalcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and 36P6D5 overexpressing PC3, 3T3 and LNCaPcells. To assay whether 36P6D5 has chemoattractant properties, parentalindicator cells are monitored for passage through the porous membranetoward a gradient of 36P6D5 conditioned media compared to control media.This assay may also be used to qualify and quantify specificneutralization of the 36P6D5 induced effect by candidate cancertherapeutic compositions. Cells can also be monitored for changes ingrowth, adhesiveness, and invasiveness in the presence and absence ofexogenously added purified 36P6D5 protein.

In another functional assay, cells stably expressing 36P6D5 can beanalyzed for their ability to form colonies in soft agar. In theseexperiments, cells used in such procedures (e.g. NIH-3T3 cells), can betransfected to stably express 36P6D5 or neo or activated-Ras (as thetest gene, the negative and the positive controls, respectively) inorder to assess the transforming capabilities of 36P6D5. Typicallyexperiments are performed in duplicate and the assays are evaluatedapproximately 4 weeks after cell plating. Where experimentalobservations demonstrate that 36P6D5 induces an increase in colonyformation relative to a negative control (e.g. neo) such resultsindicate that 36P6D5 has significant transforming capabilities.

In another functional assay, parental cells and cells expressing 36P6D5can be compared for their ability to induce cytoplasmic accumulation ofcAMP. In a typical embodiment, cells such as an LAPC xenograft (or anyof a variety of other cells such as 293T cells) can be exposed to 36P6D5and/or transfected with empty pcDNA4 HIS MAX vector or with pcDNA4 HISMAX 36P6D5. Typically, cells are starved in 1% fetal bovine serum (FBS)overnight and incubated with media alone or in the presence of asecretory molecule or 10% FBS. The cells are then lysed and analyzed forcAMP content by enzyme linked immunoassay (EIA) according to themanufacturer's recommendations (Linco Research, St Charles, Mich.).

Many genes identified as playing a role in oncogenesis function byactivating the CAMP signaling pathway. Typically, in the absence ofligand, signaling molecules are normally in an inactive state. Uponligand binding or overexpression, such molecules acquire an activeconformation and complex with G proteins. This interaction results inthe dissociation of G protein subunits and the activation of adenylatecyclase, resulting in CAMP accumulation (Birnbaumer L, Cell 1992,71:1069). Enhanced production of cAMP results in the activation ofseveral downstream signaling pathways that mediate the effect of suchmolecules. A demonstration that cells contacted by or transfected with36P6D5 leads to the accumulation of CAMP in response to FBS wouldindicate that 36P6D5 functions as a signaling molecule under theseconditions.

Example 10 In Vivo Assay for 36P6D5 Tumor Growth Promotion

The effect of the 36P6D5 protein on tumor cell growth may be evaluatedin vivo by gene overexpression in tumor-bearing mice. For example, SCIDmice can be injected SQ on each flank with 1×10⁶ of a number ofprostate, breast, colon, pancreatic, and ovarian cell lines containingtkNeo empty vector or 36P6D5. At least two strategies may be used: (1)Constitutive 36P6D5 expression under regulation of an LTR promoter, and(2) Regulated expression under control of an inducible vector system,such as ecdysone, tet, etc. Tumor volume is then monitored at theappearance of palpable tumors and followed over time to determine if36P6D5 expressing cells grow at a faster rate. Additionally, mice may beimplanted with 1×10⁵ of the same cells orthotopically to determine if36P6D5 has an effect on local growth in the target tissue (i.e.,prostate) or on the ability of the cells to metastasize, specifically tolungs, lymph nodes, liver, bone marrow, etc. In relation to prostatecancer, the effect of 36P6D5 on bone tumor formation and growth may beassessed by injecting prostate tumor cells intratibially, as describedin Example 1.

These assays are also useful to determine the 36P6D5 inhibitory effectof candidate therapeutic compositions, such as for example, 36P6D5antibodies, 36P6D5 antisense molecules and ribozymes.

Example 11 In Vitro Assay of 36P6D5 Protein Interaction

Cell lines expressing 36P6D5 can also be used to identifyprotein-protein interactions mediated by 36P6D5. The observation that36P6D5 is a secreted molecule that binds to cells (see e.g. FIG. 8)provides evidence that this molecule interacts with other proteins onthe cell surface. This interaction can be examined usingimmunoprecipitation techniques as shown by others (Hamilton B J, et al.Biochem. Biophys. Res. Commun. 1999, 261:646-51). 36P6D5 protein can beimmunoprecipitated from 36P6D5 expressing prostate cancer cell lines andexamined for protein association by western blotting. Proteininteraction may be also studied by a two yeast hybrid system, asdescribed by Shnyreva et. Al. (Shnyreva M et al, J Biol Chem. 2000. 19;275; 15498-503). These assays may also be used to analyze the effect ofpotential cancer therapeutics on 36P6D5 function.

To determine the contribution of the various domains contained withinthe 36P6D5 ORF to 36P6D5 function, 36P6D5 mutants can be generatedlacking one or more domains. Cell lines expressing mutant 36P6D5 proteinwill be evaluated for alteration in proliferation, invasion, migration,transcriptional activation and protein-protein interaction.

Example 12 Chromosomal Localization of 36P6D5

The chromosomal localization of 36P6D5 was determined using theGeneBridge4 radiation hybrid panel (Walter et al., 1994, Nat. Genetics7:22) (Research Genetics, Huntsville Ala.). The following PCR primerswere used to localize 36P6D5:

36P6D5.7 ATACCCAAAGAACGAAGCTGACAC (SEQ ID NO: 17) 36P6D5.8TACTCATCAAATATGGGCTGTTGG (SEQ ID NO: 18)The resulting mapping vector for the 93 radiation hybrid panel DNAs was:

000101110110100100100110110011111111010111111110111110100000010111011001110001101110001001011

This the mapping program which can be found at internet addresshttp://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl maps 36P6D5 tochromosome 21q22.2-22.3.

Example 13 Detecting Expression of 36P6D5 Proteins in Human Cancers

As shown in FIG. 9, a subset of cancer cells express and secrete 36P6D5protein. Conditioned media and/or cell lysates from a variety of cancercell lines representing cancers derived from prostate (LAPC4 xenograft),colon (Colo 205, CaCo-1), breast (Du4475), and pancreatic (Capan-1)tissues, as well as PC3 prostate cancer cells engineered to overexpress36P6D5 protein, were subjected to Western analysis using an anti-36P6D5murine pAb. Briefly, cells (˜25 ug total protein) and conditioned media(25 ul of neat, 0.22 uM filtered media) were solubilized in SDS-PAGEsample buffer and separated on a 10-20% SDS-PAGE gel and transferred tonitrocellulose. Blots were blocked in Tris-buffered saline (TBS)+3%non-fat milk and then probed with a 1:1,000 dilution (in TBS+0.15%Tween-20+1% milk) of serum derived from mice immunized with purified36P6D5 protein. Blots were then washed and incubated with a 1:4,000dilution of anti-mouse IgG-HRP conjugated secondary antibody. Followingwashing, anti-36P6D5 immunoreactive bands were developed and visualizedby enhanced chemiluminescence and exposure to autoradiographic film. Thespecific anti-36P6D5 immunoreactive bands representing endogenous 36P6D5protein are indicated with arrows and run approximately between 35 and40 kD. The molecular weight of 36P6D5 calculated from the amino acidsequence is 26 kD suggesting that endogenous 36P6D5 protein ispost-translationally modified, possibly by glycosylation. These resultsdemonstrate that 36P6D5 may be useful as a diagnostic and therapeutictarget for prostate, colon, breast, pancreatic and potentially otherhuman cancers.

As shown in FIG. 10, a sensitive and specific capture ELISA detects36P6D5 protein in supernatants of human cancer cell lines. A captureELISA was developed using protein G purified murine anti-36P6D5 pAb ascapture Ab and a biotinylated form of the same pAb as detection Ab.Briefly, 1 ug of purified murine anti-36P6D5 pAb was used to coat wellsof an ELISA plate. Following blocking with PBS containing 3% milk, 50 ulof conditioned media from either PC3-neo, PC3-36P6D5, or DU4475 cells orvarious amounts of purified Tag5-36P6D5 protein spiked into tissueculture media were added to wells and incubated for 2 hours at roomtemperature. Wells were washed 4× with PBS+0.05% Tween-20 (PBS-T) and 1×with PBS. Wells were then incubated for 1 hour with 3 ug/ml ofbiotinylated anti-3606D5 pAb in PBS-T+1% milk (TBS-TM, 50 ul/well) andwashed as above. Wells were then incubated with 50 ul of a 1:8,000dilution of avidin-HRP complex (Neutralite™, Southern Biotechnology,Inc) in TBS-TM for 1 hour. Following washing, wells were then developedby addition of 200 ul of TMB substrate. The reaction was stopped by theaddition of 50 ul of 1M H2SO4 and optical densities of wells were readat 450 nM. Shown is the standard curve generated using the Tag5-36P6D5protein and specific detection and quantitation of 36P6D5 present insupernatants derived from PC-3 cells overexpressing 36P6D5 andendogenous 36P6D5 protein secreted by Du4475 breast cancer cells.

FIG. 11 also shows 36P6D5 expression in human cancers. As shown in FIG.11, in a typical method for detecting expression of 36P6D5 in humancancers, cell lysates from Colon, breast and kidney cancer tissues (Ca),as well as their normal matched adjacent tissues (N) were subjected toWestern analysis using an anti-36P6D5 mouse monoclonal antibody.Briefly, tissues (˜25 ug total protein) were solubilized in SDS-PAGEsample buffer and separated on a 10-20% SDS-PAGE gel and transferred tonitrocellulose. Blots were blocked in Tris-buffered saline (TBS)+3%non-fat milk and then probed with 2 μg/ml (in TBS+0.15% Tween-20+1%milk) of purified anti-36P6D5 antibody. Blots were then washed andincubated with a 1:4,000 dilution of anti-mouse IgG-HRP conjugatedsecondary antibody. Following washing, anti-36P6D5 Immunoreactive bandswere developed and visualized by enhanced chemiluminescence and exposureto autoradiographic film. The specific anti-36P6D5 immunoreactive bandsrepresent a monomeric form of the 36P6D5 protein, which runsapproximately between 35 and 40 kD, and multimeric forms of the protein,which run approximately at 90 and 120 kD. These results demonstrate that36P6D5 may be useful as a diagnostic and therapeutic target for colon,breast, kidney and potentially other human cancers.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLE 1 predicted binding of peptides from 36P6D5 proteins to the humanMHC class I molecule HLA-A2 Start Score (Estimate of half time RankPosition Subsequence Residue Listing of disassociation)  1 7 GLLKVVFVV(SEQ ID NO: 19) 1407.5  2 97 LMGEQLGNV (SEQ ID NO: 20) 104.7  3 27LLAELIPDA (SEQ ID NO: 21) 79.6  4 188 WVFIAAKGL (SEQ ID NO: 22) 31.8  511 VVFVVFASL (SEQ ID NO: 23) 22.3  6 6 GGLLKVVFV (SEQ ID NO: 24) 21.2  7190 FIAAKGLEL (SEQ ID NO: 25) 13.5  8 89 KICFEDNLL (SEQ ID NO: 26) 10.3 9 109 INIAIVNYV (SEQ ID NO: 27) 9.8 10 181 NMKFRSSWV (SEQ ID NO: 28)9.7

TABLE 2 36P6D5 is detected in serum samples derived from cancerpatients. 36P6D5 Sample (ng/ml) normal (M) ND normal (M) ND normal (M)ND normal (M) ND normal (M) 1.31 normal (M) ND (1/6)  PC3-neo NDPC-3-36P6D5 3.38 Du4475 0.92 colon ND colon 3.08 colon 0.31 colon NDcolon 0.92 colon 0.77 colon 0.62 colon 1.23 colon 0.46 colon ND (7/10)pancreatic 0.15 pancreatic 0.38 pancreatic 29.70  pancreatic NDpancreatic 1.38 pancreatic 0.23 pancreatic ND pancreatic ND pancreatic0.38 pancreatic ND (6/10)

Clinical serum samples from and pancreatic cancer patients, and normalmale donors were screened for 36P6D5 protein using a capture ELISA asdescribed in FIG. 10. Supernatants from PC-3-36P6D5 and Du4475 cells,and from PC-3-neo cells, served as positive and negative controls,respectively for 36P6D5 protein detection. ND: not detected or belowdetection sensitivity. 36P6D5 protein was detected in 7/10 colon cancerpatients and 6/10 pancreatic cancer patients, one of which hadrelatively high levels (29.70 ng/ml), but only in 1/6 normal maledonors.

1. A method of detecting the presence of breast, colon, kidney orpancreatic cancer expressing 36P6D5 protein that comprises determiningthe level of 36P6D5 protein expressed by cells in a breast, colon,kidney or pancreatic test tissue sample from an individual, wherein the36P6D5 protein comprises SEQ ID NO:2, and comparing the level sodetermined to the level of 36P6D5 expressed in a corresponding normalbreast, colon, kidney or pancreatic tissue sample, the presence ofelevated 36P6D5 protein in the test sample relative to the normal sampleproviding an indication of the presence of such cancer in theindividual.
 2. The method of claim 1, wherein determining the level of36P6D5 protein expressed by the cells comprises contacting the cellswith an antibody that specifically binds 36P6D5 protein comprising theamino acid sequence of SEQ ID NO:2.
 3. The method of claim 2, whereinthe antibody comprises a polyclonal antibody.
 4. The method of claim 2,wherein the antibody comprises a monoclonal antibody.
 5. A method ofmonitoring 36P6D5 gene products comprising determining the status of36P6D5 gene products expressed by cells in a test breast, colon, kidneyor pancreatic tissue sample from an individual, wherein the 36P6D5 geneproduct is a protein comprising SEQ ID NO:2, and comparing the status sodetermined to the status of 36P6D5 gene products in a correspondingnormal breast, colon, kidney or pancreatic sample, the presence ofelevated status of 36P6D5 gene products in the test sample relative tothe normal sample providing an indication of dysregulated breast, colon,kidney or pancreatic cell growth within the individual.
 6. A method ofexamining a biological breast, colon, kidney or pancreatic sample forevidence of dysregulated cellular growth comprising comparing the statusof 36P6D5 in the biological breast, colon, kidney or pancreatic sampleto the status of 36P6D5 in a corresponding normal breast, colon, kidneyor pancreatic sample, wherein the status of 36P6D5 in the biologicalsample is evaluated by examining levels of 36P6D5 protein expression,wherein the 36P6D5 protein comprises SEQ ID NO:2, and wherein elevationsin the status of 36P6D5 in the biological sample are associated withdysregulated breast, colon, kidney or pancreatic cellular growth.
 7. Amethod of diagnosing the presence of breast, colon, kidney or pancreaticcancer in an individual comprising: (a) determining the level of 36P6D5protein expressed in a test breast, colon, kidney or pancreatic sampleobtained from the individual, wherein the 36P6D5 protein comprises SEQID NO:2; and (b) comparing the level so determined to the level of36P6D5 protein expressed in a comparable known normal breast, colon,kidney or pancreatic tissue sample, the presence of elevated 36P6D5protein in the test sample relative to the normal tissue sampleproviding an indication of the presence of cancer.
 8. The method ofclaim 7, wherein the sample is a colon sample.
 9. The method of claim 7,wherein determining the level of 36P6D5 protein expressed in the testsample comprises contacting the sample with an antibody thatspecifically binds 36P6D5 protein comprising the amino acid sequence ofSEQ ID NO:2.
 10. The method of claim 9, wherein the antibody comprises apolyclonal antibody.
 11. The method of claim 9, wherein the antibodycomprises a monoclonal antibody.
 12. The method of claim 1, whereinbreast tissue is examined.
 13. The method of claim 1, wherein colontissue is examined.
 14. The method of claim 1, wherein kidney tissue isexamined.
 15. The method of claim 1, wherein pancreatic tissue isexamined.
 16. The method of claim 5, wherein the status of the 36P6D5gene products is determined in a test breast tissue sample.
 17. Themethod of claim 5, wherein the status of the 36P6D5 gene products isdetermined in a test colon tissue sample.
 18. The method of claim 5,wherein the status of the 36P6D5 gene products is determined in a testkidney tissue sample.
 19. The method of claim 5, wherein the status ofthe 36P6D5 gene products is determined in a test pancreatic tissuesample.
 20. The method of claim 6, wherein a breast sample is examined.21. The method of claim 6, wherein a colon sample is examined.
 22. Themethod of claim 6, wherein a kidney sample is examined.
 23. The methodof claim 6, wherein a pancreatic sample is examined.
 24. The method ofclaim 7, wherein the sample is a breast sample.
 25. The method of claim7, wherein the sample is a kidney sample.
 26. The method of claim 7,wherein the sample is a pancreatic sample.